Nucleic acid purification with a binding matrix

ABSTRACT

The present invention relates to methods, kits, and compositions for generating purified RNA samples and purified DNA samples. In particular, the present invention provides methods for generating a purified RNA or DNA sample from a sample containing both DNA and RNA molecules using a binding matrix that preferentially binds DNA or RNA in the presence of an acidic dilution buffer, or using a binding matrix that comprises acid zeolites, as well as compositions and kits for practicing such methods.

The present application claims priority to U.S. Provisional ApplicationSer. No. 60/748,825, filed Dec. 9, 2005, which is herein incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to methods, kits, and compositions forgenerating purified RNA samples and purified DNA samples. In particular,the present invention provides methods for generating a purified RNA orDNA sample from a sample containing both DNA and RNA molecules using abinding matrix that preferentially binds DNA or RNA in the presence ofan acidic dilution buffer, or using a binding matrix that comprises acidzeolites, as well as compositions and kits for practicing such methods.

BACKGROUND OF THE INVENTION

Because of the structural similarity between DNA and RNA, previous RNApurification methods have often comprised isolating DNA and RNA togetherfrom biological sources. One commonly used method for isolating nucleicacids from cells and tissues was the “Sevag” procedure. This methodcomprises contacting a cell or tissue homogenate with phenol or amixture of phenol and chloroform, thereby denaturing proteins andprecipitating them while leaving nucleic acids in solution. This method,while still used, is hazardous, laborious and of limited utility forisolation of RNA from biological sources containing high amounts ofribonuclease (RNase), an extremely stable enzyme that degrades RNA.

An improved method for isolating intact RNA from ribonuclease-richtissues was disclosed by Chirgwin et al., Biochemistry, 18: 5924-29(1979). This method comprises exposing tissue homogenates toconcentrated guanidinium thiocyanate and 2-mercaptoethanol, therebyeliminating nucleolytic degradation of RNA by denaturing all of thecellular proteins, including ribonuclease, at a rate which exceeded therate of RNA hydrolysis by ribonuclease. Although RNA isolated in thismanner was biologically active, it was not free of contamination by DNA,protein and other cellular materials. Subsequent, often extensive,manipulation was required to further purify the RNA from other cellularcontaminants.

Silica based nucleic acid isolation techniques have been developed asalternatives to, or in addition to, the conventional isolationtechniques described above for use in isolating total RNA from at leastsome types of biological materials. For example, an RNA isolation kithas been developed that uses a glass fiber filter in a spin filterbasket and a hybrid lysis buffer/binding solution with a highconcentration of guanidine hydrochloride and a chaotropic agent toisolate total RNA from simple biological materials, such as culturedcells, blood, yeast, and bacteria (See, e.g. High Pure RNA Isolation Kitfrom Roche Diagnostics). A system for isolating total RNA from bacterialcells and tissue using a spin basket with a silica gel-based membrane,and a lysis buffer/binding solution containing guanidiniumisothiocyanate has also been developed (See, e.g. the RNeasy total RNAkit from QIAGEN Inc, Chatsworth, Calif.). Both systems described brieflyabove allow one to isolate total RNA, but the yield and purity of RNAisolated tends to be low, particularly when used to isolate RNA fromcomplex biological materials, such as plant or animal tissue.

As such, what is needed are methods, compositions, and kits that allowfor the purification of RNA from complex biological materials with highyield, while avoiding DNA contamination.

SUMMARY OF THE INVENTION

The present invention relates to methods, kits, and compositions forgenerating purified RNA samples and purified DNA samples. In particular,the present invention provides methods for generating a purified RNA orDNA sample from a sample containing both DNA and RNA molecules using abinding matrix that preferentially binds DNA or RNA in the presence ofan acidic dilution buffer, or using a binding matrix that comprises acidzeolites, as well as compositions and kits for practicing such methods.

In some embodiments, the present invention provides methods ofgenerating a purified RNA sample from an initial sample that comprisesDNA and RNA molecules, the method comprising; a) contacting the initialsample with; i) a dilution buffer with an acidic pH, and ii) a bindingmatrix (e.g. a plurality of binding particles or composition coated witha plurality of binding particles) that preferentially binds DNAmolecules in the presence of the dilution buffer, wherein the contactinggenerates a DNA-bound binding matrix; and b) separating the DNA-boundbinding matrix from the initial sample thereby generating a purified RNAsample comprising a plurality of RNA molecules.

In certain embodiments, the present invention provides methods ofgenerating a purified RNA sample, the method comprising; a) mixing aninitial sample comprising a cell suspension with a lysis buffer underconditions such that a cell lysate is generated, b) contacting the celllysate with; i) a dilution buffer with an acidic pH, and ii) a bindingmatrix that preferentially binds DNA molecules in the presence of thedilution buffer, wherein the contacting generates a DNA-bound bindingmatrix; and c) separating the DNA-bound binding matrix from the celllysate thereby generating a purified RNA sample comprising a pluralityof RNA molecules.

In certain embodiments, the binding matrix is configured to bind bothdouble stranded and single stranded DNA molecules. In other embodiments,the binding matrix is configured to not bind double stranded or singlestranded RNA molecules.

In some embodiments, the present invention provides methods ofgenerating a purified RNA sample from an initial sample that comprisesDNA and RNA molecules, the method comprising; a) contacting the initialsample with a binding matrix comprising acid zeolites such that aDNA-bound binding matrix is generated; and b) separating the DNA-boundbinding matrix from the initial sample thereby generating a purified RNAsample comprising a plurality of RNA molecules.

In certain embodiments, the present invention provides methods ofgenerating a purified sample from an initial sample that comprises DNAand target molecules comprising; a) contacting the initial sample with;i) a dilution buffer with an acidic pH, and ii) a nucleic acid bindingmatrix that preferentially binds DNA molecules in the presence of thedilution buffer, wherein the contacting generates a DNA-bound bindingmatrix; and b) separating the DNA-bound binding matrix from the initialsample thereby generating a purified sample. In some embodiments, atleast a portion of the target molecules comprise proteins. In furtherembodiments, at least a portion of said target molecules comprise lipidmolecules. In other embodiments, at least a portion of the targetmolecules comprise drug molecules. In some embodiments, the purifiedsample is substantially DNA-free.

In certain embodiments, the present invention provides methods ofgenerating a purified sample from an initial sample that comprises DNAand target molecules comprising; a) contacting the initial sample with anucleic acid binding matrix comprising acid zeolites, wherein thecontacting generates a DNA-bound binding matrix; and b) separating theDNA-bound binding matrix from the initial sample thereby generating apurified sample.

In additional embodiments, the binding matrix, whether in an acidicbuffer or not in an acidic buffer, comprises binding particles, acomposition coated with binding particles, or a solid support. Inparticular embodiments, the binding matrix is magnetic and theseparating step is performed with magnetic separation type techniques.In certain embodiments, the magnetic binding matrix comprises particlescontaining two or more magnetic cores bound to a zeolite matrix or acidzeolite matrix. In other embodiments, the particles comprising two ormore magnetic cores are covered by a zeolite or acid zeolite coating. Incertain preferred embodiments, the binding matrix comprises bindingparticles (e.g. zeolites, acid zeolites, a solid acid catalyst, etc.).In other embodiments, the binding matrix comprises a membrane (e.g.silicon membrane, zeolite or acid zeolite membrane, or combinedsilicon-zeolite or silicon-acid zeolite membrane). In certainembodiments, a zeolite membrane is formed on the surface of a siliconmembrane, or completely surrounding the silicon membrane, to form acombined silicon-zeolite membrane.

In certain embodiments, the plurality of RNA molecules comprises totalRNA from the lysed cells. In other embodiments, the plurality of RNAmolecules includes small RNA molecules (e.g. less than about 150, 120,100, 80, 70, 60, 50, or 40 bases in length). In some embodiments, themethods further comprise the step of exposing the purified RNA sample toa binding component (e.g. silicon membrane) such that an RNA-boundbinding component is generated which comprises a plurality of bound RNAmolecules. In particular embodiments, the methods further comprise thestep of washing the RNA-bound binding component (e.g. to remove saltsand impurities). In other embodiments, the methods further comprise thestep of eluting at least a portion of the bound RNA molecules from theRNA-bound binding member with a wash solution such that a purified RNApreparation is generated, wherein the purified RNA preparation comprisesa plurality of eluted RNA molecules.

In certain embodiments, the initial sample comprises a cell lysate,wherein the cell lysate comprises lysed cells, and wherein the pluralityof eluted RNA molecules are present in the purified RNA preparation at alevel of at least 5 μg of RNA per 1 million of the lysed cellsoriginally present in the sample (e.g. at least 5, 6, 7, 8, 9, 10, ormore μg of RNA per 1 million lysed cells). In particular embodiments,the amount of RNA present in the purified RNA sample and/or purified RNApreparation is at least 75% of the amount present in the originalsample. In other words, the yield is at least 75%, preferably at least85%, and more preferably at least 95%. In additional embodiments, theyield is between 80-100%, or between 95-100%.

In some embodiments, the purified RNA sample and/or purified RNApreparation are substantially DNA-free. In other embodiments, thepurified RNA sample and/or purified RNA preparation are essentiallyDNA-free. In particular embodiments, the purified RNA sample and/or thepurified RNA preparation do not contain detectable DNA when the sampleis subjected to a DNA contamination assay employing conditions asdescribed in Example 1. In other embodiments, the purified RNA sample orthe purified RNA preparation contains less than 40 discreet DNAmolecules (e.g. less than 39, 35, 30, 25, 15, 10, 5, or 0 discrete DNAmolecules per 10 ng of RNA present in the purified RNA sample orpurified RNA preparation, and which may be determined by real-time PCR).In some embodiments, the purified RNA sample or the purified RNApreparation contain less than 100 picograms of DNA, less than 75picograms of DNA, less than 50 picograms of DNA, less than 25 picogramsof DNA, or less than 10 picograms of DNA. In certain embodiments, thepurified RNA sample and/or purified RNA preparation contains less than5%, or less than 3% or less than 1%, less than 0.5%, less than 0.25%,less than 0.1%, or less than 0.05% of the mass of DNA (or number ofmolecules of DNA) present in the original sample.

In certain embodiments, the purified RNA sample or purified RNApreparation is enriched for RNA molecules compared to the initialsample. For example, RNA in a sample may be enriched (e.g., as measuredby UV absorption) about or at least about 2-fold, 3.5-fold, 5-fold,10-fold, 50-fold, 100-fold, 150-fold, 200-fold, 500-fold, 800-fold,1000-fold, 2000-fold, and all ranges therein as determined by theconcentration (e.g. ug/ml) or mass of RNA molecules relative to theconcentration or mass of total RNA molecules prior to contacting theinitial sample with the binding matrix in the acidic dilution buffer.Enrichment and/or purification may also be measured in terms of thenumber of RNA molecules relative to the number of total RNA moleculespresent in the original or initial sample. RNA molecules can be isolatedsuch that a sample is enriched (e.g., as measured by UV absorption)about or at least about 2-fold, 3.5-fold, 5-fold, 10-fold, 50-fold,100-fold, 150-fold, 200-fold, 500-fold, 800-fold, 1000-fold, 2000-fold,and all ranges therein in RNA molecules as determined by number of RNAmolecules relative to total number of RNA molecules prior to contactingthe initial sample with the binding matrix in the acidic dilutionbuffer. Enrichment and/or purification of RNAs may also be measured interms of the increase of RNA molecules relative to the number of totalRNA molecules. RNA molecules can be isolated such that the amount of RNAmolecules is increased about or at least about 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or morewith respect to the total amount of RNA in the sample before and afterisolation.

In particular embodiments, the purified RNA sample or the purified RNApreparation are DNase-free or substantially DNase free (e.g., usingAMBION's DNaseAlert™ QC System High-throughput, Fluorometric DNaseDetection Assay that sample is found to have less than 10 picograms ofDNase enzymes). In other embodiments, the initial sample is selectedfrom the following; a cell lysate, a previously purified RNA sample, anRNA control sample, a pharmaceutical drug; a protein preparation; alipid preparation; a reaction mixture where the presence of DNA isundesirable (e.g. Promega's Taq products). In some embodiments, thecontacting step is conducted under alcohol-free conditions.

In certain embodiments, the separating step comprises passing theinitial sample through a clearing column. In other embodiments, theseparating step comprises centrifuging the sample such that a pelletforms which contains the DNA-bound binding particles, and separating thepellet from the remainder of the sample.

In some embodiments, the sample is heated to a temperature of between 25and 80 degrees Celsius prior to the separating step, although higher andlower temperature may be used. In certain embodiments, the sample isheated to a temperature of between 25 and 80 degrees Celsius after thedilution buffer and binding particles have been added to the sample. Inother embodiments, the sample is heated to a temperature of between 25and 80 degrees Celsius prior to the addition of the dilution bufferand/or binding particles to the sample. In additional embodiments, thesample is heated to a temperature of at least 50 degrees, or at least 75degrees Celsius. In some embodiments, the heating is conducted for about1-15 minutes (e.g. the sample is incubated at about 75 degrees Celsiusfor about 3 minutes, although longer and shorter incubation times may beused).

In particular embodiments, the dilution buffer comprises a citratebuffer or similar buffer. In other embodiments, the dilution bufferfurther comprises about 2-4 M NaCl (or other concentrations of NaCl). Inother embodiments the dilution buffer further comprises a chaotrope(e.g. guanidinium at concentrations of 0.2M to 2M). In furtherembodiments, the sample comprises a cell lysate. In certain embodiments,the cell lysate comprises RNA molecules, DNA molecules, and non-nucleicacid cellular debris (e.g. cell walls, proteins, enzymes, or othernon-nucleic acid contents of the lysed cells). In further embodiments,the DNA-bound binding matrix (e.g. DNA-bound binding particles) alsoserve to trap or bind to the non-nucleic acid cellular debris such thatseparating the DNA-bound binding particles serves to remove asubstantial proportion of the non-nucleic acid cellular debris.

In some embodiments, the cell lysate is generated by contacting a cellsuspension comprising a plurality of cells with a lysis buffercomprising a chaotropic agent. In certain embodiments, the plurality ofcells are selected from: human cells, mouse cells, plant cells, bacteriacells, transformed cells, or other type of cell. In additionalembodiments, the chaotropic agent is selected from the group consistingof sodium perchlorate, guanidinium hydrochloride, guanidiniumisothiocyanate, guanidinium thiocyanate, sodium iodide, potassiumiodide, and combinations thereof. In particular embodiments, thechaotropic agent is present in a concentration from about 0.1M to about10.0M, or similar concentrations. In further embodiments, the lysisbuffer further comprises a reducing agent (e.g. beta-mercaptoethanol).

In preferred embodiments, the binding matrix comprises bindingparticles. In certain embodiments, the binding particles include, butare not limited to, silica gel, sol gel, glass, powdered glass, quartz,alumina, zeolites, acid zeolites, Fe₂O₃-zeolite, Silica-Fe₂O₃-zeoliteparticles (e.g., MagneSil®-zeolite particles), titanium dioxide, andzirconium dioxide. In some embodiments, the binding particles comprisesilicon. In particular embodiments, the binding particles comprisetectosilicates. In preferred embodiments, the binding particles comprisezeolite particles. In preferred embodiments, the binding particles aresynthetic molecular sieve zeolites, preferably Type 13X, or acid zeolitederivatives of zeolite 13X. In other preferred embodiments, the bindingparticles are synthetic molecular sieve zeolites, preferably Type 3A, oracid zeolite derivatives of Type 3A. In other preferred embodiments, thebinding particles are synthetic molecular sieve zeolites, preferably amixture of Type 3A and Type 13X, or mixtures of their acid zeolitederivatives.

In particular embodiments, the particle size is from 0.1 μm to 100 μm,but is not limited to such sizes. In other embodiments, the bindingparticles are less than about 15 μm or less than 10 μm is size. In someembodiments, the binding particles have a pore size between 3-15 Å(e.g., 3, 5, 7, 8, 10, 12, or 14 Å). In preferred embodiments, the poresize is about 10 Å. However, the present invention is not limited tosuch sizes.

In some embodiments, the dilution buffer has a pH of 6.5 or less or 6.0or less. In other embodiments, the dilution buffer has a pH between 3.0and 5.5. In particular embodiments, the DNA molecules in the samplecomprise genomic DNA molecules. In further embodiments, the clearingagent further comprises 1-2 M NaCl.

In certain embodiments, the present invention provides kits forgenerating a purified RNA sample comprising; a) a dilution buffer withan acidic pH; and b) a binding matrix, wherein the binding matrix isconfigured to preferentially bind DNA molecules in the presence of thedilution buffer. In certain embodiments, the binding matrix comprisesbinding particles (e.g. membrane coated with binding particles or aclearing agent solution comprising binding particles).

In particular embodiments, the present invention provides kits forgenerating a purified RNA sample comprising; a) a binding matrixconfigured to preferentially bind DNA molecules in the presence of adilution buffer with an acidic pH; and b) a kit component selected fromthe following list: i) the dilution buffer; ii) an insert component,wherein the insert component comprises written instructions for usingthe binding matrix to remove DNA molecules from a sample containing bothDNA and RNA molecules; iii) a lysis buffer, wherein the lysis buffercomprises a chaotropic agent; iv) a wash solution; v) a clearing column;and vi) a binding column. In certain embodiments, the kits furthercomprise a second, third, fourth, fifth, or sixth component selectedfrom the list of kit components. In some embodiments, the kits comprisecontrol samples (e.g. negative and/or positive controls). In particularembodiments, the binding matrix comprises binding particles.

In some embodiments, the present invention provides kits for generatinga purified RNA sample comprising; a) a dilution buffer with an acidicpH; and b) an insert component, wherein the insert component compriseswritten instructions for using a binding matrix to remove DNA moleculesfrom a sample containing the dilution buffer and both DNA and RNAmolecules, wherein the binding matrix preferentially binds DNA moleculesin the presence of the dilution buffer.

In certain embodiments, the present invention provides compositionscomprising: i) an initial sample comprising DNA and RNA molecules, andii) a binding matrix, wherein the binding matrix is bound to 10 timesthe number of DNA molecules as RNA molecules.

In particular embodiments, the present invention provides compositionscomprising; a) a dilution buffer with an acidic pH; and b) a bindingmatrix that preferentially binds DNA molecules in the presence of thedilution buffer. In further embodiments, the compositions furthercomprise a sample comprising DNA and RNA molecules. In otherembodiments, the compositions further comprise a lysis buffer, whereinthe lysis buffer comprises a chaotropic agent.

In some embodiments, the present invention provides compositionscomprising; a) zeolite particles present at a concentration of about0.1-1.0 g/ml; and b) a NaCl solution present at a concentration of about1-3M. In certain embodiments, the zeolite particles are present at aconcentration of about 0.5 g/ml (e.g. 0.2-0.8 g/ml). In otherembodiments, the NaCl solution is present at a concentration of about 2M. In particular embodiments, the compositions further comprise achelating agent. In additional embodiments, the chelating agentcomprises ethylene diamine tetra-acetic acid (EDTA) present at aconcentration of about 0.1 mM to 10 mM with a pH of about 8.0. Inpreferred embodiments, the zeolite particles are configured topreferentially bind DNA in the presence of a buffer with an acidic pH.In other embodiments, the zeolite particles comprise Type 13X molecularsieve zeolites, or acid zeolite derivatives of Type 13X. In otherembodiments, the zeolite particles comprise Type 3A molecular sievezeolites or acid zeolite derivatives of Type 3A zeolites. In otherembodiments, the zeolite particles comprise a mixture of Type 3A andType 13X molecular sieve zeolites, or mixtures of acid zeolitederivatives thereof.

In particular embodiments, the present invention provides compositioncomprising about 35-55% by weight (e.g., 35% . . . 40% . . . 47% . . .55%) zeolites (e.g., 13× zeolites), 1.5-2.5 M NaCl (e.g., 2.0M NaCl),and 0.05 to 1.5 mM EDTA (e.g, 0.1 mM EDTA).

In certain embodiments, the RNA purification methods of the presentinvention are used prior to RT-PCR or quantitative RT-PCR, or otherprocedures where it is preferred that DNA not be present. In someembodiments, the RNA purification methods are used for research,diagnostics, preparation of therapeutics, quality control monitoring, orany other application known or later discovered where it is desired tocollect, purify, analyze, detect, and/or characterize nucleic acidmolecules (e.g. RNA molecules).

In other embodiments, the present invention provides methods ofgenerating a purified DNA sample from an initial sample that comprisesDNA and RNA molecules, the method comprising; a) contacting the initialsample with; i) a dilution buffer with an acidic pH, and ii) a bindingmatrix (e.g. a plurality of binding particles or composition coated witha plurality of binding particles) that preferentially binds RNAmolecules in the presence of the dilution buffer, wherein the contactinggenerates a RNA-bound binding matrix; and b) separating the RNA-boundbinding matrix from the initial sample thereby generating a purified DNAsample comprising a plurality of DNA molecules. In preferredembodiments, the binding matrix that preferentially binds RNA moleculesin the presence of the dilution buffer comprises titanium oxide.

In further embodiments, the present invention provides methods ofgenerating a purified DNA sample, the method comprising; a) mixing aninitial sample comprising a cell suspension with a lysis buffer underconditions such that a cell lysate is generated, b) contacting the celllysate with; i) a dilution buffer with an acidic pH, and ii) a bindingmatrix that preferentially binds RNA molecules in the presence of thedilution buffer, wherein the contacting generates a RNA-bound bindingmatrix; and c) separating the RNA-bound binding matrix from the celllysate thereby generating a purified DNA sample comprising a pluralityof DNA molecules.

In some embodiments, the methods further comprise the step of exposingthe purified DNA sample to a binding component such that a DNA-boundbinding component is generated which comprises a plurality of bound DNAmolecules. In particular embodiments, the methods further comprise thestep of washing the DNA-bound binding component (e.g. to remove saltsand impurities). In other embodiments, the methods further comprise thestep of eluting at least a portion of the bound DNA molecules from theDNA-bound binding member with a wash solution such that a purified DNApreparation is generated, wherein the purified DNA preparation comprisesa plurality of eluted DNA molecules.

In certain embodiments, the sample comprises a cell lysate, wherein thecell lysate comprises lysed cells, and wherein the plurality of elutedDNA molecules are present in the purified DNA preparation at a level ofat least 5 μg of DNA per 1 million of the lysed cells originally presentin the sample (e.g. at least 5, 6, 7, 8, 9, 10, or more μg of DNA per 1million lysed cells). In particular embodiments, the amount of DNApresent in the purified DNA sample and/or purified DNA preparation is atleast 75% of the amount present in the original sample. In other words,the yield is at least 75%, preferably at least 85%, and more preferablyat least 95%. In additional embodiments, the yield is between 80-100%,or between 95-100%.

In some embodiments, the purified DNA sample and/or purified DNApreparation are substantially RNA-free. In other embodiments, thepurified DNA sample and/or purified DNA preparation are essentiallyRNA-free. In particular embodiments, the purified DNA sample and/or thepurified DNA preparation do not contain detectable RNA when the sampleis subjected to a RNA contamination assay employing conditions asdescribed in Example 9. In other embodiments, the purified DNA sample orthe purified DNA preparation contains less than 40 discreet RNAmolecules (e.g. less than 39, 35, 30, 25, 15, 10, 5, or 0 discrete DNAmolecules per 10 ug of DNA as determined by real-time PCR). In someembodiments, the purified DNA sample or the purified DNA preparationcontain less than 100 picograms of RNA, less than 75 picograms of RNA,less than 50 picograms of RNA, less than 25 picograms of RNA, or lessthan 10 picograms of RNA. In certain embodiments, the purified DNAsample and/or purified DNA preparation contains less than 5%, or lessthan 3% or less than 1%, less than 0.5%, less than 0.1%, or less than0.05% of the mass of RNA present in the original sample.

In particular embodiments, the present invention provides kits forgenerating a purified DNA sample comprising; a) a binding matrixconfigured to preferentially bind RNA molecules (e.g. titanium oxide) inthe presence of a dilution buffer with an acidic pH; and b) a kitcomponent selected from the following list: i) the dilution buffer; ii)an insert component, wherein the insert component comprises writteninstructions for using the binding matrix to remove RNA molecules from asample containing both DNA and RNA molecules; iii) a lysis buffer,wherein the lysis buffer comprises a chaotropic agent; iv) a washsolution; v) a clearing column; and vi) a binding column. In certainembodiments, the kits further comprise a second, third, fourth, fifth,or sixth component selected from the list of kit components. In someembodiments, the kits comprise control samples (e.g. negative and/orpositive controls). In particular embodiments, the binding matrixcomprises binding particles.

In some embodiments, the present invention provides kits for generatinga purified RNA sample comprising; a) a binding matrix comprising acidzeolites configured to preferentially bind DNA molecules; and b) atleast one kit component selected from the following list: i) a dilutionbuffer; ii) an insert component, wherein the insert component compriseswritten instructions for using the binding matrix to remove DNAmolecules from an initial sample containing both DNA and RNA molecules;iii) a lysis buffer, wherein the lysis buffer comprises a chaotropicagent; iv) a wash solution; v) a clearing column; and vi) a bindingcolumn.

In particular embodiments, the present invention provides an articlecomprising a membrane, wherein the membrane comprises a binding matrix(e.g. titanium oxide, zeolite particles, acid zeolites, etc.) configuredto preferentially bind DNA or RNA in an acidic buffer. In someembodiments, the present invention provides an article comprising amembrane, wherein the membrane comprises zeolites, or acid zeolites. Insome embodiments, the present invention provides an article comprising amembrane, wherein the membrane comprises a silica membrane coated withzeolite, or acid zeolite derivatives thereof (e.g., a standard siliconmembrane coated on all surfaces with zeolites). In some embodiments, thepresent invention provides an article comprising a membrane, wherein themembrane comprises zeolite particles. In certain embodiments, themembrane is configured to be inserted into a binding column (e.g, abinding column, or 96 well plate, generally used to purify RNA or DNAsamples with the aid of a centrifuge and/or vacuum system to pull liquidthrough the membrane). In other embodiments, the membrane furthercomprises silica (e.g. the membrane material is made of silica and/orsilica that is bound to zeolite particles). In further embodiments, themembrane is approximately circular in shape. In certain embodiments, themembrane is less than about 4, or 3 or 2 centimeters in length ordiameter, or wherein said membrane has a diameter between about 7 andabout 11 millimeters or between 8 and 10 millimeters. In someembodiments, the membrane is between about 5 millimeters and 30millimeters (e.g. 5 mm, 7 mm, 9 mm, 9.1 mm, 9.5 mm, 10 mm, 14 mm, 14.4mm, 25 mm, 26 mm, 26.7 mm, 26.9 mm or 30 mm). In some embodiments, themembrane is less than about 3, or 2 or 1 millimeters in thickness. Inparticular embodiments, the membrane is composed entirely or nearlyentirely of zeolites (e.g. at least 95%, 98%, or 99% of the membrane iscomposed of zeolites).

In particular embodiments, the present invention provides DNA-bindingmembranes comprising zeolites or acid zeolites. In certain embodiments,the membranes are configured to be inserted into a binding column. Insome embodiments, the DNA-binding membrane further comprises silica. Inother embodiments, the membrane is approximately circular in shape (e.g.elliptical, circular, etc.). In further embodiments, the membrane isless than about 4, or less than about 3, or less than about 2centimeters in diameter, or wherein said membrane has a diameter betweenabout 5 and about 35 millimeters or between 8 and 10 millimeters. Insome embodiments, the membrane is between about 5 millimeters and 30millimeters (e.g. 5 mm, 7 mm, 9 mm, 9.1 mm, 9.5 mm, 10 mm, 14 mm, 14.4mm, 25 mm, 26 mm, 26.7 mm, 26.9 mm or 30 mm). In certain embodiments,the membrane is less than about 3 millimeters in thickness (e.g., lessthan about 2 millimeter or less than 1 millimeter, or between about 2.9and 1.2 millimeters in thickness). In particular embodiments, themembrane is composed entirely or nearly entirely of acid zeolites (e.g.at least 90%, 95% or 98% of the membrane is composed of acid zeolites).

In some embodiments, the present invention provides a DNA-bindingmembrane comprising: i) a silicon membrane comprising a top surface anda bottom surface, and ii) zeolites, wherein the zeolites are disposed onthe top surface, on the bottom surface, or on both the top and bottomsurfaces. In certain embodiments, at least 75%, 80%, 90%, or 99% of thetop surface is covered with the zeolites. In other embodiments, at least75%, 90%, 90%, or 99% of the bottom surface is covered with thezeolites. In particular embodiments, the membrane is between about 5-30millimeters in diameter. In other embodiments, the membrane is betweenabout 8-10 millimeters in diameter. In further embodiments, the membraneis approximately circular in shape (e.g. circular, elliptical, nearlycircular, etc.). In some embodiments, the membrane is between about 5millimeters and 30 millimeters (e.g. 5 mm, 7 mm, 9 mm, 9.1 mm, 9.5 mm,10 mm, 14 mm, 14.4 mm, 25 mm, 26 mm, 26.7 mm, 26.9 mm or 30 mm). Inother embodiments, the membrane is between less than about 4 centimetersin diameter. In further embodiments, the membrane is approximatelycircular in shape.

Membranes according to the present invention could be used withcommercially available sample preparation devices such as single-columncentrifuge tubes and multi-well plates. Commercially available tubesfrom companies such as Eppendorf, Corning and Becton Dickinson's Falcon®brand include 0.5 mL, 0.65 mL, 11.0 mL, 1.2 mL, 1.5 mL, 1.7 mL, 2.0 mL,2.1 mL, 2.2 mL, 5 mL, 8 mL, 10 mL, 13 mL, 15 mL, 20 mL, 50 mL, 175 mLand 225 mL capacity centrifuge tubes. The diameters of these tubes varydepending on design but are typically in the range of about 3.0 mm toabout 61 mm. For example a BD Falcon™ Conical Centrifuge tube, 15 mLcapacity has an approximate outer diameter (“O.D.”) of 17 mm, the 50 mLcapacity tube has an O.D. of 30 mm, and the 175 mL and 225 mL tubes havean O.D. of 61 mm. Molecular Research Center Inc. provides polypropylenecentrifuge tubes with a 5 mL capacity (13 mm O.D.), 8 mL capacity (13 mmO.D.), 13 mL capacity (17 mm O.D.) and 20 mL capacity (21 mm O.D.).CryoStor™ Vials sold by Denville Scientific Inc. (Metuchen, N.J.) with avolume capacity of 0.65 mL, 1.7 mL and 2.0 mL all have an O.D. of 10 mm.Sample preparation devices are typically made from plastics such asclear polypropylene (PP) or polyethylene terephthalate (PET) but can beconstructed with any material that would not substantially interferewith the sample. The internal diameter of a tube depends on the tubeconfiguration, construction materials and manufacturing tolerances. Forexample, centrifuge tubes: 1) from Corning (Corning, N.Y.) include a 50mL capacity tube which has a diameter of 26.9 mm; a 15 mL capacity witha diameter of 14.4 mm; and a 2.0 mL capacity tube with a diameter of 9.5mm; 2) from Fisher Scientific (Hampton, N.H.) include a 50 mL capacitytube with a diameter of 26.7 mm; and 3) from Eppendorf, include a 1.5 mLcapacity tube with a diameter of 9.0 mm-9.1 mm, and a 2.0 mL capacitytube with a diameter of 9.0 mm.

The membranes could also be used in conjunction with well-plates forsimultaneous preparation of, for example, 6, 8, 10, 12, 24, 48, 96, 384and 1586 samples. Wells in commercially available plates are typicallysquared or rounded in shape and are typically in a uniform patternacross the plate. Many manufacturers of well plates use a standarddimensions calculated from center to center of the wells in allow theplates to be utilized on different robotic instruments. 96 well plates,for example, typically measure 9 mm from center to center of the wells.The internal dimensions will also depend on the configuration of thewells, construction materials and manufacturing tolerances. Theselection of membrane size is dependent on the internaldimensions/configuration of these preparation devices, constructionmaterials and manufacturing tolerances that vary from manufacturer tomanufacturer.

DESCRIPTION OF THE FIGURES

FIG. 1A shows an ethidium bromide stained gel of human cell lysatesamples purified as described in Example 1. FIG. 1B shows the results ofa DNA contamination assay as described in Example 1 were exposed toRNase digestion prior to loading on the gel.

FIG. 2A shows an ethidium bromide stained gel of the cow spleen celllysate samples purified as described in Example 2. FIG. 2B shows theresults of a DNA contamination assay where the samples described inExample 2 were exposed to RNase digestion prior to loading on the gel.

FIG. 3A shows an ethidium bromide stained gel of the transformed E. colicell lysate samples purified as described in Example 3. FIG. 3B showsthe results of a DNA contamination assay where the samples described inExample 3 were exposed to RNase digestion prior to loading on the gel.

FIG. 4A shows an ethidium bromide stained gel of the rat liver celllysate samples purified as described in Example 4. FIG. 4B shows theresults of a DNA contamination assay where the samples described inExample 4 were exposed to RNase digestion prior to loading on the gel.

FIG. 5A shows an ethidium bromide stained gel of the bovine liver celllysate samples purified as described in Example 6. FIG. 5B shows theresults of a DNA contamination assay where the samples described inExample 6 were exposed to RNase digestion prior to loading on the gel.

FIG. 6A shows an ethidium bromide stained gel of the human blood celllysate samples purified as described in Example 7. FIG. 6B shows theresults of a DNA contamination assay where the samples described inExample 7 were exposed to RNase digestion prior to loading on the gel.

FIG. 7A shows an ethidium bromide stained gel of the cow spleen celllysate samples purified as described in Example 8. FIG. 7B shows theresults of an assay used to determine if DNA and/or RNA is present invarious samples as described in Example 8.

FIG. 7C shows the results of a DNA contamination assay described inExample 8.

FIG. 8 shows the results of an RNase digestion assay performed asdescribed in Example 9.

FIGS. 9A and 9B show the results of Example 11 which describes the useof zeolite membranes, silica-zeolite composite membranes, andsilica-zeolite composite in solution to remove DNA from RNA in tissuesample purifications.

FIGS. 10A, 10B, and 10C show the results of Example 12, which describesmethods used to screen various zeolites to determine their nucleic acidpurification properties.

FIGS. 11A, 11B, and 11C show the results of Example 14, which describesthe use of Fe₂O₃ particles coated with zeolite and MagneSil® particlescoated with zeolite to purify RNA from lysate.

FIG. 12 shows the results from Example 15, which describes the use ofmagnetic zeolite particles to generate a purified RNA sample.

FIG. 13 shows the results of Example 16, which describes the effect ofguanidine on RNA purification with zeolites.

FIGS. 14A and 14B show the results of Example 17, which describesvarious pH ranges for citrate buffer that are effective for purifyingRNA with zeolites.

FIG. 15 shows the results of Example 18, which describes the results ofusing various pH ranges for citrate buffers on the selective removal ofDNA from mixtures of RNA and DNA.

FIG. 16 shows the results of Example 19, which describes the results ofremoving DNA from a solution containing a mixture of protein and DNA.

FIG. 17 shows the results of Example 20, which describes the use ofzeolites and acetate buffer for generating purified RNA samples.

FIG. 18 shows the results of Example 21, which describes the effect ofheat on RNA purification with zeolites.

FIG. 19 shows the results of Example 22, which describes the use of heatat different temperatures to improve the binding of DNA to zeolites.

FIG. 20 shows the results of Example 23, which describes the binding ofzeolite 13X to a mixture of double stranded 35 bp DNA, single stranded35 base DNA, single stranded 30 base DNA, 25 bp double stranded RNA, 25base single stranded RNA and 21 base single stranded RNA, and elutionsof the bound oligonucleotides.

FIG. 21 shows the results of Example 24, which describes the binding ofzeolite to a mixture of: genomic DNA double stranded 35 bp DNA, singlestranded 35 base DNA, single stranded 30 base DNA, 25 bp double strandedRNA, 25 base single stranded RNA and 20 base single stranded RNA.

FIG. 22 shows the results of Example 25, which describes thequantitation of genomic DNA contamination in total RNA samples byquantitative PCR (qPCR) and the quantitation of specific mRNAs byquantitative RT-PCR (qRT-PCR).

DEFINITIONS

To facilitate an understanding of the invention, a number of terms aredefined below.

As used herein, the phrase “a sample that comprises DNA and RNAmolecules” is used to refer to any type of sample, such as biological orenvironmental samples, that includes a detectable quantity of both DNAand RNA molecules. Examples of such samples include, but are not limitedto, a cell lysate, a previously purified RNA sample that still containsdetectable quantities of DNA molecules, a RNA control sample, productsof an in vitro transcription/translation reaction, or products of an invitro translation reaction.

As used herein, the phrase “a sample that comprises DNA and a targetmolecule” is used to refer to any type of sample, such as a biologicalor environmental sample, that includes a detectable quantity of DNA andthe target molecule. Examples of such target molecules in a sampleinclude, but are not limited to a pharmaceutical drug preparation, aprotein preparation, a lipid preparation, any reaction mixture where thepresence of DNA is undesirable.

As used herein, a dilution buffer is said to have “an acidic pH” whenthe pH of the dilution buffer is less than 7.0. In preferredembodiments, the pH is less than 6.0, and more preferably is betweenabout 3.0 and 5.3, or between about 4.0 and 5.0, although lower pHvalues are contemplated.

As used herein, the phrase “binding matrix that preferentially binds DNAmolecules,” refers to any type of substrate, whether porous ornon-porous, that, in the presence of a buffer with an acidic pH and asample containing both DNA and RNA molecules, binds readily to the DNAmolecules present in the sample, but that has limited or no binding tothe RNA molecules present in the sample. In certain embodiments, suchbinding matrices bind DNA molecules 10 times as readily as they bindingRNA molecules (e.g. a 10/1 DNA to RNA ratio), while in otherembodiments, the binding matrices bind DNA molecules 20 times, 50 times,or 100 times as readily as they bind RNA molecules (e.g. 20/1, 50/1, or100/1 DNA to RNA ratio). Examples of such binding matrices include, butare not limited to, zeolite membranes, acid zeolite membranes, bindingparticles, a composition coated with binding particles (e.g. silicamembrane coated with binding particles, magnetic particles coated withbinding particles, silica membrane coated with zeolite, magneticparticles coated with zeolite, glass bubbles, zirconia, or aluminacoated with binding particles, glass bubbles, zirconia, or aluminacoated with zeolite), and a solid support.

As used herein, the phrase “binding particles that preferentially bindDNA molecules” refers to any type of small particles (e.g. micrometerrange) that are porous or non-porous and that have the ability to serveas a binding matrix that preferentially binds DNA molecules. Examples ofsuch binding particles include, but are not limited to, silica gel, solgel, glass, powdered glass, quartz, alumina, zeolite particles,zirconium dioxide, and tectosilicates. In preferred embodiments, thebinding particles comprise synthetic molecular sieve zeolites,preferably Type 13X or Type 3A.

As used herein, the phrase “binding matrix that preferentially binds RNAmolecules,” refers to any type of substrate, whether porous ornon-porous, that, in the presence of a buffer with an acidic pH and asample containing both DNA and RNA molecules, binds readily to the RNAmolecules present in the sample, but that has limited or no binding tothe DNA molecules present in the sample. In certain embodiments, suchbinding matrices bind RNA molecules 10 times as readily as they bindingDNA molecules (e.g. a 10/1 RNA to DNA ratio), while in otherembodiments, the binding matrices bind RNA molecules 20 times, 50 times,or 100 times as readily as they bind DNA molecules (e.g. 20/1, 50/1, or100/1 RNA to DNA ratio). Examples of such binding matrices include, butare not limited to, titanium oxide particles.

As used herein, the phrase “binding particles that preferentially bindRNA molecules” refers to any type of small particles (e.g. micrometerrange) that are porous or non-porous and that have the ability to serveas a binding matrix that preferentially binds RNA molecules.

As used herein, a purified RNA sample or purified RNA preparation isconsidered “substantially DNA-free” when, of all the nucleic acidpresent in the sample, less than 1.0% of the total mass of nucleic acidis DNA (i.e. at least 99.1% of the total mass of the nucleic acidpresent is RNA). The mass of nucleic acid present may be determined bymass spectrometry or other methods used to determine mass.

As used, herein, a purified RNA sample or purified RNA preparation isconsidered “essentially DNA-free” when, of all the nucleic acid presentin the sample, less than 0.25% of the total mass of nucleic acid is DNA(i.e. at least 99.76% of the total mass of the nucleic acid present isRNA). The mass of nucleic acid present may be determined by massspectrometry or other methods used to determine mass.

As used herein, a purified DNA sample or purified DNA preparation isconsidered “substantially RNA-free” when, of all the nucleic acidpresent in the sample, less than 1.0% of the total mass of nucleic acidis RNA (i.e. at least 99.1% of the total mass of the nucleic acidpresent is DNA). The mass of nucleic acid present may be determined bymass spectrometry, UV absorption methods, or other methods used toquantitate nucleic acid molecules.

As used, herein, a purified DNA sample or purified DNA preparation isconsidered “essentially RNA-free” when, of all the nucleic acid presentin the sample, less than 0.25% of the total mass of nucleic acid is RNA(i.e. at least 99.76% of the total mass of the nucleic acid present isDNA). The mass of nucleic acid present may be determined by massspectrometry, UV absorption methods, or other methods used to quantitatenucleic acid molecules.

DESCRIPTION OF THE INVENTION

The present invention relates to methods, kits, and compositions forgenerating purified RNA samples and purified DNA samples. In particular,the present invention provides methods for generating a purified RNA orDNA sample from a sample containing both DNA and RNA molecules using abinding matrix that preferentially binds DNA or RNA in the presence ofan acidic dilution buffer, or using a binding matrix that comprises acidzeolites, as well as compositions and kits for practicing such methods.

I. Nucleic Acid Purification with a Binding Matrix

The compositions and methods of the present invention allow purified RNAand DNA samples to be prepared that contain very low levels of DNA orRNA contamination, yet provide a high yield of RNA or DNA to be purifiedfrom the original sample. For example, as shown in Example 6 below, thepresent invention provides a level of RNA purity superior than can beachieved with standard TRIzol reagent purification. This surprisinglevel of purification, without the need for time consuming and extensiveprocessing of samples satisfies the need in the art for highly purifiedRNA samples. For example, procedures in the art such as RT-PCR orquantitative RT-PCR, or other procedures that benefit from highlypurified RNA, should greatly benefit from the present invention.

RNA or DNA may be isolated and purified according to the presentinvention from any type of nucleic acid preparation, biological sample,cell lysate, tissue culture, tissue homogenate, or any other type ofsample that contains both DNA and RNA molecules. Exemplary samplesinclude, but are not limited to, blood, urine, endocrine fluid, tissues,cells, and lysates of tissues or cells. In certain preferredembodiments, the sample comprises a cell lysate.

Cell lysates may be prepared, for example, by methods known in the art.Generally, a cell suspension, tissue, organ, plant leaves, or othersource of cells is mixed with a lysis buffer comprising a chaotropicsalt in order to rupture the cells. The mixture is rapidly homogenized,using, for example, a hand held homogenizer or an automatic homogenizer,such as a Waring blender, a Polytron tissue homogenizer, or the like.

The present invention is not limited to any specific type of lysisbuffer. Preferably, the lysis buffer contains a chaotropic salt, presentin about a 1-5 M concentration. Chaotropic salts are salts of chaotropicions. Such salts are highly soluble in aqueous solutions. The chaotropicions provided by such salts, at sufficiently high concentration inaqueous solutions of proteins or nucleic acids, cause proteins tounfold, nucleic acids to lose secondary structure or, in the case ofdouble-stranded nucleic acids, to melt. Chaotropic ions include, forexample, guanidinium, iodide, perchlorate and trichloroacetate. Inpreferred embodiments, the chaotropic ion is the guanidinium ion.Examples of chaotropic salts include, for example, guanidinehydrochloride, guanidine thiocyanate (which is sometimes referred to asguanidine isothiocyanate), sodium iodide, sodium perchlorate, and sodiumtrichloroacetate. Preferred are the guanidinium salts, more preferablyguanidine hydrochloride or guanidine thiocyanate, but most preferablyguanidine thiocyanate (GTC).

In certain embodiments that employ an acidic dilution buffer, after thecells are lysed (if the original sample contains cells) the samplecontaining both DNA and RNA molecules is contacted with a dilutionbuffer with an acidic pH, as well as with a binding matrix (e.g. bindingparticles) that preferentially binds DNA. The binding matrix is added tothe sample at a level such that substantially all of the DNA (or all ofthe RNA) present in the sample can be bound by the binding particles tocreate a DNA-bound binding matrix (e.g. DNA-bound binding particles), orto create a RNA-bound binding matrix. In certain embodiments wherebinding particles are employed, the binding particles are provided atabout 0.01-1.0 grams per milliliter of sample. Additional detailsregarding binding matrices are provided in part II below.

With regard to the dilution buffer, this solution may be used to dilutethe original sample. The dilution buffer preferably contains water,salt, and a buffer and, in certain embodiments, has a pH of less than7.0 (e.g. 6.9 or less). While the present invention is not limited toany particular acidic dilution buffer, the dilution buffer preferablycomprises a citrate buffer composed of sodium citrate, sodium chloride,and water, at an acidic pH. The pH may be adjusted below 7.0 by using aconcentrated acid such as HCl.

In certain embodiments, a heating step is employed. In such embodiments,the original sample containing DNA and RNA molecules (e.g. cell lysate)is heated to a temperature between 30-80 degrees Celsius (e.g. in ahybridization oven) prior to, or after, the addition of the dilutionbuffer and binding matrix.

The sample (now containing a binding matrix, and in certain embodiments,an acidic dilution buffer) is then subjected to a process that removesthe DNA-bound binding matrix (or RNA-bound binding matrix), as well asany cellular debris that may also be present in the sample. Any type ofmethod may be used, including methods known in the art for separatingcellular debris. For example, the sample may be subjected tocentrifugation such that a pellet forms that can then be removed. TheDNA-bound binding matrix (or RNA-bound binding matrix), such as bindingparticles, and cellular debris (if present) can also be removed byfiltering. In other embodiments, the DNA-bound binding matrix (orRNA-bound binding matrix), such as magnetic binding particles, andcellular debris (if present) can also be removed by magnetic separation(see, e.g., U.S. Pat. Nos. 6,673,631 and 6,194,562, both of which areherein incorporated by reference). For example, a clearing columncontaining a membrane (e.g, paper or silica membrane) may be used. Alsofor example, if the binding matrix is a membrane coated with bindingparticles, the membrane can simply be removed from the sample. Theresult of this filtration, centrifugation, magnetic separation or otherremoval techniques is a purified RNA sample (or purified DNA sample).

In certain embodiments, such as those relating to RNA purification, thepurified RNA sample is further processed to further purify the RNAmolecules away from any remaining contaminants. In certain preferredembodiments, the purified RNA sample is mixed with an alcohol (e.g.,isopropanol) and then exposed to an RNA binding component (e.g. silicamembrane, poly-T coated surface, etc.) such that RNA binds to the RNAbinding component. In certain preferred embodiments, a binding column isemployed (e.g. with a silica membrane). A description of such bindingcolumns is provided in U.S. Pat. No. 6,218,531, herein incorporated byreference in its entirety. In certain embodiments, the RNA bindingcomponent is washed with a wash solution to remove salts and otherdebris. The RNA can be eluted from the RNA binding component usingstandard methods. For example, nuclease free water may be employed toelute the bound RNA molecules such that a purified RNA preparation isgenerated.

II. Binding Matrices

The present invention is not limited by the type of binding matricesthat are employed. Instead, any type of binding matrices thatpreferentially binds DNA molecules (e.g. zeolites, acid zeolites, etc.),or that preferentially binds RNA molecules (e.g. titanium oxide) in thepresence of an acidic dilution buffer may be employed. In preferredembodiments, the binding matrix comprises binding particles. Preferablythe binding particles are micrometer is size (e.g. between 0.1 and 1000μm in size). In preferred embodiments, the binding particles are lessthan about 20 μm in size and in particularly preferred embodiments, thebinding particles are less than 10 μm in size (e.g. less than 10 μm inany planar dimension).

The binding matrices of the present invention may be porous ornon-porous. Preferably the binding matrices contain pores that arebetween about 1 and 20 Å in size. In particularly preferred embodiments,the pores are about 9-11 Å in size (e.g. about 10 Å).

The binding matrices may be composed of any type of material. Examplesof such materials include, but are not limited to, silica gel, sol gel,glass, powdered glass, quartz, alumina, zeolite particles, titaniumdioxide, zirconium dioxide, and tectosilicates. Additional bindingparticles are described in U.S. Pat. No. 5,155,018 to Gillespie et al.and U.S. Pat. No. 6,180,778 to Bastian et al., both of which are hereinincorporated by reference in their entireties for all purposes. Incertain preferred embodiments, the binding matrices are zeoliteparticles (e.g. zeolite particles with pores).

The zeolites are framework silicates and are generally composed ofinterlocking tetrahedrons of SiO₄ and AlO₄. In general, the ratio of(Si+Al)/O in a zeolite equals approximately ½. The alumino-silicatestructure is negatively charged and attracts the positive cations thatreside within. Unlike most other tectosilicates, zeolites have largevacant spaces or cages in their structures that allow space for largecations such as sodium, potassium, barium and calcium and evenrelatively large molecules and cation groups such as water, ammonia,carbonate ions, nitrate ions, and other molecules. In certain zeolites,the spaces are interconnected and form long wide channels of varyingsizes depending on the mineral. These channels allow the easy movementof the resident ions and molecules into and out of the structure.Zeolites are characterized by their ability to lose and absorb waterwithout damage to their crystal structures.

There are currently about 45 natural minerals that are recognizedmembers of the zeolite group. Industrially speaking, the term zeoliteincludes natural silicate zeolites, synthetic materials and phosphateminerals that have a zeolite like structure. The complexity of thiscombined group is extensive with over 120 structural variations.Exemplary members of the zeolite group include the following: TheAnalcime Family: Analcime, Pollucite, Wairakite, Bellbergite, Bikitaite,Boggsite, and Brewsterite; The Chabazite Family: Chabazite,Willhendersonite, Cowlesite, Dachiardite, Edingtonite, Epistilbite,Erionite, Faujasite, and Ferrierite; The Gismondine Family: Amicite,Garronite, Gismondine, Gobbinsite, Gmelinite, Gonnardite, andGoosecreekite; The Harmotome Family: Harmotome, Phillipsite, andWellsite; The Heulandite Family: Clinoptilolite, Heulandite, Laumontite,Levyne, Mazzite, Merlinoite, Montesommaite, and Mordenite; The NatroliteFamily: Mesolite, Natrolite, Scolecite, Offretite, Paranatrolite,Paulingite, and Perlialite; The Stilbite Family: Barrerite, Stilbite,Stellerite, Thomsonite, Tschernichite, and Yugawaralite.

There are also many different synthetic zeolites. One type of syntheticzeolite is the molecular sieve zeolites. Examples of these zeolitesinclude Type 3A (3 Å pore size), Type 4A (4 Å pore size), Type 5A (5 Åpore size) and Type 13X (10 Å pore size). In preferred embodiments, thebinding particles of the present invention comprise Type 13X zeolites.In other preferred embodiments, the binding particles of the presentinvention comprise Type 3A zeolites.

In certain embodiments, the binding matrix comprises acid zeolites.Under certain acidic conditions, the cation in a zeolite may be replacedby a proton, producing an “acid zeolite” (Armengol, Corma Garcia andPrimo, (1995) Applied Catalysis A General, 126, p. 391-399; see alsoCliment et al., Applied Catalysts A: General 130, 5-12, 1995, both ofwhich are herein incorporated by reference.) Acid zeolites are generallyconsidered crystalline alumino-silicates with a high internal surfacearea (approx. 1000 m²/g), containing acidic hydroxyl (O—H) groupslocated in nanometer micropores (See, e.g., J. M. Thomas, Scient. Am.266(IV)(1992)82, pgs. 112-118, herein incorporated by reference). Thedegree of acidity of the acid zeolite and the reaction conditions hasbeen shown to affect Freidel-Crafts alkylation of benzene and toluenewith cinnamyl alcohol (Armengol et al (1995) above). In certainembodiments, the acid zeolite is ZSM-5 or similar acid zeolite.

Acid zeolites can be generated from zeolites by high temperaturereactions. For example, a zeolite sample can be calcined to remove theorganic cation. Zeolites can be placed inside a tube furnace on top of afrit in the middle of the tube, and spread out to maximize the surfacearea. One can then fit a ground glass elbow at each end, one attached toa nitrogen cylinder and the other immersed in a beaker of water, toregulate the flow of nitrogen gas. The zeolite can be slowly heated inthe tube to 500° C. in increments of 50 to 100° C., with water vaporbeing released, then in increments of 100 to 500° C. This heating can beconducted for two hours after reaching temperature, where thetetrapropylammonium bromide will decompose to tripropylamine, propylene,and water. Any sodium ions remaining in the zeolite will now be ionexchanged for protons to fully convert the zeolite to the acid form.

The zeolites thus may be converted to their acid zeolite matricesthrough the use of an acidic buffer and method of treatment, asdetermined using screening methodology, such as that described inExample 12 below. The treatment may include the use of guanidine as anadditive to the acidic buffer, or a heat treatment step, in the methodof obtaining an acid zeolite particle, membrane, or magnetic acidzeolite particle. After the treatment, the acidic buffer is removed,leaving the acid zeolite matrix. The acid zeolite matrix is then used topurify RNA from a mixture of RNA and DNA in a liquid solution, therebyremoving substantially all of the DNA. The liquid solution does not needto be at an acidic pH for the acid zeolite matrix to preferentiallyremove DNA from the sample. The sample may also be a mixture of proteinand DNA, as described in Example 19, or may be a mixture of DNA and apharmaceutical drug.

Zeolites have many minerals that have similar cage-like frameworkstructures or have similar properties and/or are associated withzeolites, but that are not specifically classified as zeolites. Examplesof these include the phosphates: kehoeite, pahasapaite and tiptopite;and the silicates: hsianghualite, lovdarite, viseite, partheite,prehnite, roggianite, apophyllite, gyrolite, maricopaite, okenite,tacharanite and tobermorite.

Under certain acidic conditions, the cation may be replaced by a proton,producing an “acid zeolite” (Armengol, Corma Garcia and Primo, (1995)Applied Catalysis A General, 126, p. 391-399). The degree of acidity ofthe acid zeolite and the reaction conditions has been shown to affectFreidel-Crafts alkylation of benzene and toluene with cinnamyl alcohol(Armengol et al (1995) above).

Another class of mineral that the binding particles of the presentinvention may be composed of are the tectosilicates. This subclass isoften called the “Framework Silicates” because its structure is composedof interconnected tetrahedrons going outward in all directions formingan intricate framework analogous to the framework of large building. Inthis subclass all the oxygens are shared with other tetrahedrons givinga silicon to oxygen ratio of approximately 1:2.

In order to determine if a certain type of matrix may serve as a usefulbinding matrix in the methods of the present invention, one can screenthe candidate matrices in methods similar to Example 12 or Examples 1-7below or as described in the following exemplary protocol. First a celllysate is prepared by homogenizing a tissue or cell sample in LysisBuffer (e.g., 4M guanidine thiocyanate, 10 mM Tris, pH 7.5, withbeta-mercaptoethanol) with a rotor/stator homogenizer. The lysate isplaced in two separate tubes, with one of the tubes being left as alysate (control) and one of the tubes further processed as follows.

An acidic dilution buffer (e.g., 3M NaCl, 0.3M Na₃ citrate•2H₂O, 0.2%SDS, 0.0009% Blue dye (FD&C Blue #1), adjusted to a final pH of 4.0 withconcentrated hydrochloric acid (HCl)) is then added to the one tubecontaining the lysate to be further processed at a ratio of 2:1 (v/v).The sample in the tube is then mixed by inversion until homogeneous. Thetube is incubated at about 50-70° C. in a hybridization oven for about 3minutes and then placed at ambient temperature. The candidate bindingmatrix, which may be binding particles, are then weighed and added tothe tube at a ratio of about 0.25 g per ml of lysate volume. Thismixture is then shaken vigorously and poured into a clearing column(e.g. Promega PureYield™ Clearing Column), nested in a 50 ml collectiontube. The column is then centrifuged at 2,000×g, at 23° C. for about 10minutes. If the candidate binding matrix is able to serve as asuccessful binding matrix according to the present invention, the lysateshould contain RNA and will be captured in the 50 ml collection tube,while the sample debris, and DNA bound binding matrix will be capturedby the clearing column membrane and can be discarded with the clearingcolumn.

The lysate is further processed as follows prior to performing theassays to determine if the candidate binding matrix was in factsuccessful at preferentially binding the DNA. Isopropanol is added tothe cleared lysate at a volume equal to the added dilution buffervolume. The sample is mixed and then immediately applied to a bindingcolumn (e.g. Promega PureYield™ Binding Column), attached to a vacuummanifold with a vacuum of approximately 15 in. Hg which will cause thesample to pass through the column leaving RNA present in the samplebound to the binding column membrane. The membrane is then washed twicewith 10 ml and then 20 ml of Wash Solution (e.g., 60 mM potassiumacetate, 10 mM Tris, pH 7.5, 60% ethanol) to remove impurities andsalts. The membrane is then vacuum dried for 3 minutes on a vacuummanifold. The binding column is then transferred to 50 ml collectiontubes for elution with nuclease-free water. The column assembly is thencentrifuged (e.g, using a swinging bucket rotor) at 2,000×g for 2minutes to collect RNA that may be bound to the membrane. The elutionsteps are then repeated, with an additional 1 ml of nuclease-free water.

The purified sample, as well as the unprocessed original lysate sample,are then analyzed for both RNA and DNA content. In particular, thesesamples are analyzed by gel electrophoresis on a native, 1% agarose, 1×TBE gel, stained with ethidium bromide (e.g. as described in theExamples below). This gel indicates the total amount of nucleic acid ineach sample. A second gel is then run to determine what percent, if any,of the nucleic acid visualized in the first gel is DNA contaminationinstead of the desired RNA. Samples are digested with RNase (e.g.Promega's RNase ONE™ Ribonuclease, Cat.# M4265). The digestions areincubated at 37° C. for 1 hour and then held at 4° C. The digests arethen analyzed by electrophoresis on a native, 1% agarose, 1× TBE gel,stained with ethidium bromide. The results of this second gel indicatethe amount of DNA that is present in the samples.

The success of the candidate binding matrix can be determined byreviewing each of these gels. Preferably, the second gel contains lessthan about 1% DNA compared to the total amount of RNA present asvisualized in the first gel. Particularly preferred binding matrices areidentified when there is no easily visualized DNA present in the treatedsample lane in the second gel, yet there is a substantial amount ofnucleic acid in the first gel compared to the untreated lysate.

The above exemplary screening procedure may also be employed to screencandidate acidic dilution buffers. For example, one could use a bindingagent known to preferentially bind DNA in the protocol described above,but substitute in a candidate acidic dilution buffer (e.g. to testdifferent buffers as well as different pH's). In this regard, the twogels that result could be analyzed to determine if a given candidatedilution buffer is suitable for use with the methods of the presentinvention.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: N (normal); M (molar); mM (millimolar); μM(micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg(micrograms); ng (nanograms); l or L (liters); ml (milliliters); μl(microliters); Xg (time gravity); and C (degrees Centigrade).

Example 1 Isolation of Total RNA From Human Cells

This example describes the purification of total RNA from human HEK293Tcells. Human HEK293T cells were cultured in monolayers under standardconditions in flasks. The cells were trypsinized, washed and harvestedby centrifugation. Cell pellets were flash frozen in a dry ice/ethanolbath. The cell pellets were stored at −70° C. until use.

A tube containing a pellet of ˜6×10⁸ cells was placed on dry ice. Twelveml of 4° C. Lysis Buffer (4M guanidine thiocyanate, 10 mM Tris, pH 7.5,with beta-mercaptoethanol added separately to a final concentration of0.974% (v/v)) was immediately added to the tube, after it wastransferred to ice. The cell pellet was disrupted and the cells werelysed by homogenization with a Tissue-Tearor™ rotor/stator homogenizer(Biospec Products, Inc., Bartlesville, Okla.). The sample washomogenized 2-3 times until the pellet was no longer visible. In thisExample, 5 ml of a previously prepared lysate was combined with thefreshly prepared lysate, to increase the amount of lysate available. Theadded lysate was prepared using the same procedure, stored at −70° C.and then thawed at 4° C. before use. The combined lysate was dividedinto four 2 ml and four 1 ml aliquots to isolate RNA from ˜1×10⁸ and˜5×10⁷ cells per prep, respectively. An additional 1 ml of Lysis Bufferwas added to the 1 ml samples. Plastic, 15 ml, screw capped tubes wereused. The samples were divided into two sets, designated “1MIX” and“2MIX”, to test a variation on mixing in this Example.

The 1MIX samples were processed as follows. Four ml of Dilution Buffer(3M NaCl, 0.36M Na₃ citrate•2H₂O, 0.0009% Blue dye (FD&C Blue #1),adjusted to a final pH of 4.0 with concentrated hydrochloric acid (HCl))was added to each quadruplicate sample of 2 ml of lysate, withoutmixing. One ml of Clearing Agent (2M NaCl, 0.1 mM EDTA (pH 8.0), 0.45g/ml Molecular Sieves, type 13X (zeolite) binding particles) was addedto each sample. The samples were then mixed by inversion and vortexing,until homogeneous. These four samples were designated “1MIX”.

The 2MIX samples were processed as follows. Four ml of Dilution Buffer(3M NaCl, 0.36M Na₃ citrate•2H₂O, 0.0009% Blue dye (FD&C Blue #1),adjusted to a final pH of 4.0 with concentrated hydrochloric acid (HCl))was added to each quadruplicate 2 ml of lysate and mixed by inversionand vortexing, until homogeneous. One ml of Clearing Agent (2M NaCl, 0.1mM EDTA (pH 8.0), 0.45 g/ml Molecular Sieves, type 13X (zeolite)) wasadded to each sample and mixed by inversion and vortexing, untilhomogeneous. These four samples were designated “2MIX” for “2 Mixes”.

The “1 MIX” and “2MIX” samples were incubated at 70° C. in ahybridization oven for 5 minutes and then placed at ambient temperature(23° C.). The remaining steps were performed at ambient temperature (23°C.). Each mixture was applied to a Promega clearing column (PromegaPureYield™ Clearing Column; which has a cellulose membrane) in a 50 mlcollection tube and centrifuged at 2,000×g for 10 minutes in a swingingbucket rotor to clear the lysates. The cleared lysates, containing RNA,were captured in the collection tubes. The sample debris, bindingparticles and DNA were captured by the clearing column membrane and werediscarded with the clearing columns.

Four ml of isopropanol was added to each cleared lysate and mixed byswirling the tube. Each mixture was applied to a Promega binding column(Promega PureYield™ Binding Column; which has a silica membrane),attached to a vacuum manifold. A vacuum of approximately 15 in. Hg wasapplied to the columns. Each sample passed through the column, leavingthe RNA bound to the binding column membrane. The membranes were washedtwice with 20 ml and then 10 ml of Wash Solution (60 mM potassiumacetate, 10 mM Tris, pH 7.5, 60% ethanol) to remove impurities andsalts. The membranes were vacuum dried for 3 minutes on the vacuummanifold. The binding columns were transferred to 50 ml collection tubesfor elution. One ml of nuclease-free water was applied to each membraneand incubated for 2 minutes at 23° C. The column assemblies werecentrifuged, using a swinging bucket rotor, at 2,000×g for 2 minutes tocollect the purified total RNA. The elution steps were repeated, with anadditional 1 ml of nuclease-free water. The second eluates werecollected in fresh 50 ml tubes. The purified total RNA samples wereanalyzed by spectrophotometry and agarose gel analysis. Total RNA yieldswere determined by absorbance at 260 nm. The results are shown in Table1 and FIGS. 1A and 1B. TABLE 1 Total RNA Yield from Tissue Culture CellsTotal RNA Yield Total RNA Yield Total RNA Yield Sample ID Elution 1 (μg)Elution 2 (μg) Elution 1 + 2 (μg) 1E8 1MIX 1 808.9 198.0 1006.9 1E8 1MIX2 909.2 155.3 1064.5 1E8 2MIX 1 899.9 188.7 1088.6 1E8 2MIX 2 929.5199.7 1129.2 5E7 1MIX 1 473.5 76.1 549.6 5E7 1MIX 2 445.2 60.8 506.0 5E72MIX 1 444.0 42.4 486.4 5E7 2MIX 2 450.6 43.5 494.1

FIG. 1A show the RNA samples analyzed by electrophoresis on a native, 1%agarose, 1× TBE gel, stained with ethidium bromide. Ten μl of eachsample was loaded per lane. The image in FIG. 1A was collected using anAlpha Innotech FluorChem™ Imaging System. Lanes 1-8 show the Elution 1results and Lanes 9-16 show the Elution 2 results. Lanes 1 and 9 contain1E8 1MIX 1; Lanes 2 and 10 contain 1E8 1MIX 2; Lanes 3 and 11 contain1E8 2MIX 1; Lanes 4 and 12 contain 1E8 2MIX 2; Lanes 5 and 13 contain5E7 1MIX 1; Lanes 6 and 14 contain 5E7 1MIX 2; Lanes 7 and 15 contain5E7 2MIX 1; Lanes 8 and 16 contain 5E7 2MIX 2; Lane M contains Promega's1 kb DNA Ladder (Cat.# G5711), 10,000 bp, 8,000 bp, 6,000 bp, 5,000 bp,4,000 bp, 3,000 bp, 2,500 bp, 2,000 bp, 1,500 bp, 1,000 bp, 750 bp, 500bp, 253 bp and 250 bp. Representative size markers are indicated in thisfigure. The undenatured 28S ribosomal RNA, 18S ribosomal RNA and smallRNAs are indicated by arrows in this figure.

FIG. 1B depicts the results of a DNA Contamination Assay. The purifiedtotal RNA samples were digested with Promega's RNase ONE™ Ribonuclease(Cat.# M4265). A 15 μl digestion mix, consisting of 3 μl of 10× ReactionBuffer, 9 μl of Nuclease-Free Water and 3 μl of RNase ONE™ Ribonuclease(5-10 u/μl) was mixed with 15 μl of each total RNA sample. Thedigestions were incubated at 37° C. for 1 hour and then held at 4° C.until analyzed. The digests were analyzed by electrophoresis on anative, 1% agarose, 1× TBE gel, stained with ethidium bromide. Ten μl ofeach reaction was loaded per lane. The image in FIG. 1B was collectedusing an Alpha Innotech FluorChem™ Imaging System. The lanes are thesame as indicated for FIG. 1A. Representative size markers areindicated. Note that the size markers were affected by diffusion of saltfrom the digests.

Example 2 Isolation of Total RNA from Cow Spleen Cells

This example describes the isolation of total RNA from cow spleen cells.A cow spleen lysate was prepared by homogenizing frozen tissue in 4° C.Lysis Buffer (4M guanidine thiocyanate, 10 mM Tris, pH 7.5, withbeta-mercaptoethanol added separately to a final concentration of 0.974%(v/v)) with a PRO 200 rotor/stator homogenizer (PRO Scientific, Inc.,Oxford, Conn.). The lysate was prepared at a concentration of 150 mg/ml(wet weight) and was stored in 10 ml aliquots at −70° C., until use. Thefrozen lysate was thawed at 4° C. and pooled. Two 2 ml aliquots weredispensed into two plastic, 50 ml, screw capped tubes. These two sampleswere designated “DB4-” to indicate that the lysate was not furtherprocessed. The remainder of the cow spleen lysate was rehomogenized toeliminate the particulates that were observed. This rehomogenized lysatewas dispensed into eight 2 ml aliquots. Two of the samples weredesignated “DB4 R” for “rehomogenized lysate” (2 ml). Additional 4° C.Lysis Buffer was added to select samples, such that the final lysatevolumes were increased in duplicate to 3 ml, 4 ml and 5 ml per tube.Each tube contained 300 mg of cow spleen, regardless of the final lysateconcentration. Dilution Buffer (3M NaCl, 0.3M Na₃ citrate•2H₂O, 0.2%SDS, 0.0009% Blue dye (FD&C Blue #1), adjusted to a final pH of 4.0 withconcentrated hydrochloric acid (HCl)) was added to each lysate at aratio of 2:1 (v/v) (i.e. 4 ml, 6 ml, 8 ml or 1 0 ml). The samples weremixed by inversion 3-4 times, until homogeneous. Each tube was incubatedat 70° C. in a hybridization oven for 3 minutes and then placed atambient temperature (23° C.). Molecular Sieves, Type 13X, <10 μm powder(zeolite) (binding particles) was weighed and added to each tube at aratio of 0.25 g per ml of lysate volume (i.e. 0.5 g, 0.75 g, 1.0 g or1.25 g). Each mixture was shaken vigorously and poured into a Promegaclearing column (Promega PureYield™ Clearing Column), nested in a 50 mlcollection tube. The columns were centrifuged in a swinging bucket rotorat 2,000×g, 23° C. for 10 minutes. The cleared lysates, containing RNA,were captured in 50 ml collection tubes. The sample debris, bindingparticles and DNA were captured by the clearing column membrane and werediscarded with the clearing columns.

The following steps were performed at ambient temperature (23° C.).Isopropanol was added to each cleared lysate at a volume equal to theadded Dilution Buffer volume (4 ml, 6 ml, 8 ml or 10 ml). Each samplewas mixed and immediately applied to a Promega binding column (PromegaPureYield™ Binding Column), attached to a vacuum manifold. A vacuum ofapproximately 15 in. Hg was applied to the columns. Each sample passedthrough the column, leaving the RNA bound to the binding columnmembrane. The membranes were washed twice with 10 ml and then 20 ml ofWash Solution (60 mM potassium acetate, 10 mM Tris, pH 7.5, 60% ethanol)to remove impurities and salts. The membranes were vacuum dried for 3minutes on the vacuum manifold. The binding columns were transferred to50 ml collection tubes for elution. One ml of nuclease-free water wasapplied to each membrane and incubated for 1 minute at 23° C. The columnassemblies were centrifuged, using a swinging bucket rotor, at 2,000×gfor 2 minutes to collect the purified total RNA. The elution steps wererepeated, with an additional 1 ml of nuclease-free water. The secondeluates were collected in fresh 50 ml tubes. The purified total RNAsamples were analyzed by spectrophotometry and agarose gel analysis.Total RNA yields were determined by absorbance at 260 nm. The resultsare shown in Table 2 and FIGS. 2A and 2B. TABLE 2 Total RNA Yield from300 mg Cow Spleen Total RNA Total RNA Total RNA Yield Yield YieldElution Sample ID Elution 1 (μg) Elution 2 (μg) 1 + 2 (μg) DB4 - 1 (2 mlLysate) 329.6 101.7 431.3 DB4 - 2 (2 ml Lysate) 343.9 97.3 441.2 DB4 R 1(2 ml Lysate) 340.9 79.8 420.7 DB4 R 2 (2 ml Lysate) 332.1 100.4 432.5 3ml 1 341.4 75.8 417.2 3 ml 2 371.9 53.1 425.0 4 ml 1 350.4 72.5 422.9 4ml 2 334.6 86.5 421.1 5 ml 1 348.8 94.3 443.1 5 ml 2 376.2 66.9 443.1

FIG. 2A shows a gel analysis of purified total RNA from cow spleen. RNAsamples were analyzed by electrophoresis on a native, 1% agarose, 1× TBEgel, stained with ethidium bromide. Ten μl of each sample was loaded perlane. The image was collected using an Alpha Innotech FluorChem™ ImagingSystem. Lanes 1-10 show the Elution 1 results and Lanes 11-20 show theElution 2 results. Lanes 1, 11: DB4—1 (2 ml Lysate); Lanes 2, 12: DB4—2(2 ml Lysate); Lanes 3, 13: DB4 R1; Lanes 4, 14: DB4 R 2; Lanes 5, 15: 3ml Lysate 1; Lanes 6, 16: 3 ml Lysate 2; Lanes 7, 17: 4 ml Lysate 1;Lanes 8, 18: 4 ml Lysate 2; Lanes 9, 19: 5 ml Lysate 1; Lanes 10, 20: 5ml Lysate 2. The undenatured 28S ribosomal RNA, 18S ribosomal RNA andsmall RNAs are indicated by arrows.

FIG. 2B shows the results of a DNA contamination assay. Purified totalRNA samples were digested with Promega's RNase ONE™ Ribonuclease (Cat.#M4265). A 20 μl digestion mix, consisting of 4 μl of 10× ReactionBuffer, 14 μl of Nuclease-Free Water and 2 μl of RNase ONE™ Ribonuclease(5-10 u/μl) was mixed with 20 μl of each total RNA sample. Thedigestions were incubated at 37° C. for 1 hour and then held at 4° C.,until analyzed. The digests were analyzed by electrophoresis on anative, 1% agarose, 1× TBE gel, stained with ethidium bromide. Ten μl ofeach reaction was loaded per lane. The image was collected using anAlpha Innotech FluorChem™ Imaging System. Lanes 1-10 show the Elution 1results and Lanes 11-20 show the Elution 2 results. Lanes 1, 11: DB4—1(2 ml Lysate); Lanes 2, 12: DB4—2 (2 ml Lysate); Lanes 3, 13: DB4 R 1;Lanes 4, 14: DB4 R2; Lanes 5, 15: 3 ml Lysate 1; Lanes 6, 16: 3 mlLysate 2; Lanes 7, 17: 4 ml Lysate 1; Lanes 8, 18: 4 ml Lysate 2; Lanes9, 19: 5 ml Lysate 1; Lanes 10, 20: 5 ml Lysate 2. Partially degradedtotal RNA from the RNase ONE™ Ribonuclease digestion is indicated inlanes 1-10 by an arrow.

Example 3 Isolation of Total RNA from Transformed E. coli

This example describes the isolation of total RNA from transformed E.coli cells. E. coli strain JM109, transformed with the high copy vectorpGEM®-3Zf(+), was grown at 37° C. in Luria Bertani (LB) culture mediumwith 100 μg/ml ampicillin. An overnight culture was used to inoculatefresh medium and the cells were grown to an optical density of 0.69 at600 nm, corresponding to approximately 1.7×10⁸ cells/ml. The cells wereharvested by centrifugation in tubes containing 40 ml aliquots ofculture. The cell pellets were drained of medium and flash frozen inindividual tubes in a dry ice/ethanol bath. The cell pellets, containingapproximately 6.8×10⁹ E. coli cells each, were stored at −70° C. untiluse.

Tubes containing the frozen cell pellets were thawed on ice. The pelletswere gently resuspended by mixing. A freshly prepared lysozyme solution(10 mM Tris, 1 mM EDTA, (pH 7.5), 0.4 mg/ml lysozyme) was added at anamount of 0.7 ml per tube. This mixture was incubated at ambienttemperature (23° C.) for 5 minutes to digest away the bacterial cellwall.

Two experiments were performed, isolating total RNA from ˜6.8×10⁹ and˜1×10¹⁰ cells. In the first experiment, three tubes of cells (˜6.8×10⁹cells per tube) were thawed on ice for approximately 20 minutes anddigested with lysozyme as described above. A total of 4 ml of 4° C.Lysis Buffer (4M guanidine thiocyanate, 10 mM Tris, pH 7.5, withbeta-mercaptoethanol added separately to a final concentration of 0.974%(v/v)) was added to these three tubes of treated cells and combined. Thetreated cells were lysed by vigorously vortexing the tubes. The preparedlysate was dispensed into three 2 ml samples of 6.8×10⁹ cells perisolation. Plastic 15 ml, screw capped tubes were used. One ml ofClearing Agent (2M NaCl, 0.1 mM EDTA (pH 8.0), 0.45 g/ml MolecularSieves, type 13X (zeolite) type binding particles) was added to eachtube containing 2 ml of lysate and mixed by vortexing. Four ml ofDilution Buffer (3M NaCl, 0.36M Na₃ citrate•2H₂O, 0.0009% Blue dye (FD&CBlue #1), adjusted to a final pH of 4.0 with concentrated hydrochloricacid (HCl)) was added to each tube. The mixture was shaken and vortexedto homogeneity. The tubes were incubated at 70° C. in a hybridizationoven for 5 minutes and then cooled at ambient temperature (23° C.).

In the second experiment, three tubes of cells (˜6.8×10⁹ cells per tube)were thawed on ice for approximately 10 minutes and digested withlysozyme as described above. A total of 2 ml of 4° C. Lysis Buffer (4Mguanidine thiocyanate, 10 mM Tris, pH 7.5, with beta-mercaptoethanoladded separately to 1%) was added to three tubes of treated cells andcombined. The treated cells were lysed by vigorously vortexing thetubes. The prepared lysate was dispensed into two 2 ml samples of˜1×10¹⁰ cells each for RNA isolation. Plastic, 15 ml, screw capped tubeswere used. Four ml of Dilution Buffer (3M NaCl, 0.36M Na₃ citrate•2H₂O,0.0009% Blue dye (FD&C Blue #1), adjusted to a final pH of 4.0 withconcentrated hydrochloric acid (HCl)) was added to each tube containing2 ml of lysate and mixed by inversion and vortexing, until homogeneous.One ml of Clearing Agent (2M NaCl, 0.1 mM EDTA (pH 8.0), 0.45 g/mlMolecular Sieves, type 13X (zeolite) type binding particles) was addedto each tube and mixed by inversion and vortexing, until homogeneous.The tubes were incubated at 70° C. in a hybridization oven for 5 minutesand then cooled at ambient temperature (23° C.).

The following steps were performed at ambient temperature (23° C.) forboth experiments. The mixtures were applied to Promega clearing columns(Promega PureYield™ Clearing Column) in 50 ml collection tubes andcentrifuged in a swinging bucket rotor at 2,000×g for 10 minutes toclear the lysates. The clearing agent, cellular debris and DNA werecaptured by the clearing column membrane and were discarded. The clearedlysate, containing RNA, was captured in the collection tube. Four ml ofisopropanol was added to each tube of cleared lysate and mixed byswirling the tube. Each mixture was applied to a Promega binding column(Promega PureYield™ Binding Column), attached to a vacuum manifold. Avacuum of approximately 15 in. Hg was applied to the columns. Eachsample passed through the column, leaving the RNA bound to the bindingcolumn membrane. The membranes were washed twice with 20 ml and then 10ml of Wash Solution (60 mM potassium acetate, 10 mM Tris, pH 7.5, 60%ethanol) to remove impurities and salts. The membranes were vacuum driedfor 3 minutes on the vacuum manifold. The binding columns weretransferred to 50 ml collection tubes for elution. One ml ofnuclease-free water was applied to each membrane and incubated for 1-2minutes at 23° C. The column assemblies were centrifuged, using aswinging bucket rotor, at 2,000×g for 2 minutes to collect the purifiedtotal RNA. The elution steps were repeated, with an additional 1 ml ofnuclease-free water. The second eluates were collected in fresh 50 mltubes. The purified total RNA samples were analyzed by spectrophotometryand agarose gel analysis. Total RNA yields were determined by absorbanceat 260 nm. The results are shown in Table 3 and FIGS. 3A and 3B. TABLE 3Total RNA Yield from Transformed E. coli Cells Total RNA Total RNA YieldYield Total RNA Yield Sample ID Elution 1 (μg) Elution 2 (μg) Elution1 + 2 (μg) EC 1 6.8 × 10⁹ cells 640.8 156.6 797.4 EC 1 6.8 × 10⁹ cells639.8 164.1 803.9 EC 3 6.8 × 10⁹ cells 587.1 158.0 745.1 1 1 × 10¹⁰cells 743.4 204.0 947.4 2 1 × 10¹⁰ cells 822.0 189.6 1011.6

FIG. 3A shows a gel analysis of purified total RNA from transformed E.coli cells. RNA samples were analyzed by electrophoresis on a native, 1%agarose, 1× TBE gel, stained with ethidium bromide. Ten μl of eachsample was loaded per lane. The image was collected using an AlphaInnotech FluorChem™ Imaging System. Panel 1 shows the Elution 1 resultsand Panel 2 shows the Elution 2 results. Lanes 1, 6: EC 1 6.8×10⁹ cells;Lanes 2, 7: EC 16.8×10⁹ cells; Lanes 3, 8: EC 3 6.8×10⁹ cells; Lanes 4,9: 1×10¹⁰ cells; Lanes 5, 10: 2 1×10¹⁰ cells. Lane P: Promega'spGEM®-3Zf(+) Vector (circular) (Cat.# P2271), 2 μg. Lane M: Promega's 1kb DNA Ladder (Cat.# G5711), 10,000 bp, 8,000 bp, 6,000 bp, 5,000 bp,4,000 bp, 3,000 bp, 2,500 bp, 2,000 bp, 1,500 bp, 1,000 bp, 750 bp, 500bp, 253 bp and 250 bp. Representative size markers are indicated. Theundenatured 23S ribosomal RNA, 16S ribosomal RNA and small RNAs areindicated by arrows.

FIG. 3B shows a DNA Contamination Assay of Total RNA Purified from E.coli. Purified total RNA samples were digested with Promega's RNase ONE™Ribonuclease (Cat.# M4265). A 15 μl digestion mix, consisting of 3 μl of10× Reaction Buffer, 9111 of Nuclease-Free Water and 3 μl of RNase ONE™Ribonuclease (5-10 u/μl) was mixed with 15 μl of each total RNA sample.The digestions were incubated at 37° C. for 1 hour and then held at 4°C., until analyzed. The digests were analyzed by electrophoresis on anative, 1% agarose, 1× TBE gel, stained with ethidium bromide. Ten μl ofeach reaction was loaded per lane. The image was collected using anAlpha Innotech FluorChem™ Imaging System. Panel 1 shows the Elution 1results and Panel 2 shows the Elution 2 results. Lanes 1, 6: EC 16.8×10⁹ cells; Lanes 2, 7: EC 1 6.8×10⁹ cells; Lanes 3, 8: EC 3 6.8×10⁹cells; Lanes 4, 9: 1 1×10¹⁰ cells; Lanes 5, 10: 2 1×10¹⁰ cells. Lane P:Promega's pGEM®-3Zf (+) Vector (circular) (Cat.# P2271), 2 μg. Lane G:Control samples containing genomic DNA. Lane M: Promega's 1 kb DNALadder (Cat.# G5711), 10,000 bp, 8,000 bp, 6,000 bp, 5,000 bp, 4,000 bp,3,000 bp, 2,500 bp, 2,000 bp, 1,500 bp, 1,000 bp, 750 bp, 500 bp, 253 bpand 250 bp. Representative size markers are indicated.

Example 4 Isolation of Total RNA from Rat Liver

This example describes the isolation of total RNA from rat liver. A ratliver lysate was prepared by homogenizing frozen tissue in 4° C. LysisBuffer (4M guanidine thiocyanate, 10 mM Tris, pH 7.5, withbeta-mercaptoethanol added separately to a final concentration of 0.974%(v/v)) with a PRO200 rotor/stator homogenizer (PRO Scientific, Inc.,Oxford, Conn.). The lysate was prepared at a concentration of 150 mg/ml(wet weight) and was stored in 10 ml aliquots at −70° C., until use.

The frozen lysate was thawed at 4° C., pooled and dispensed into 2 mlaliquots. Plastic, 15 ml, screw capped tubes were used. Four ml ofDilution Buffer (3M NaCl, 0.3M Na₃ citrate•2H₂O, 0.2% SDS, 0.0009% Bluedye (FD&C Blue #1), adjusted to a final pH of 4.0 with concentratedhydrochloric acid (HCl)) was added to each sample and mixed by inversion3-4 times, until homogeneous. The tubes were incubated at 70° C. in ahybridization oven for 3 minutes and then placed at ambient temperature(23° C.).

Molecular Sieves, Type 13X, powder (<10 μm) (zeolite) (binding agent)was weighed and added to each tube. Duplicate samples were isolated foreach sample set: the amount of zeolite was varied from 0.25 g, 0.5 g,0.75 g, 1.0 g, and 1.5 g per tube. Each mixture was shaken vigorouslyand immediately poured into a Promega clearing column (PromegaPureYield™ Clearing Column), nested in a 50 ml collection tube. Thecolumns were centrifuged in a swinging bucket rotor at 2,000×g, 23° C.for 10 minutes. The cleared lysates, containing RNA, were captured in 50ml collection tubes. The cellular debris, binding particles and DNA werecaptured by the clearing column membrane and were discarded with theclearing columns. Two additional 0.5 g samples were cleared by highspeed centrifugation, instead of using clearing columns. For these twosamples (designated with an “S”), each mixture was vigorously shaken andpoured into a 50 ml, polypropylene, Sepcor® centrifuge tube with cap(Labcor Products, Inc., Frederick, Md.). The lysates were cleared bycentrifugation in a fixed angle rotor at 33,000×g, 20° C. for 10minutes. The cleared lysates, containing RNA, were transferred to freshtubes. The pellets, containing sample debris, DNA and binding particles,were discarded.

The following steps were performed at ambient temperature (23° C.). Fourml of isopropanol was added to each duplicate set of cleared lysates andmixed by inversion. Each mixture was applied to a Promega binding column(Promega PureYield™ Binding Column), attached to a vacuum manifold. Avacuum of approximately 15 in. Hg was applied to the columns. Eachsample passed through the column, leaving the RNA bound to the bindingcolumn membrane. The membranes were washed twice with 10 ml and then 20ml of Wash Solution (60 mM potassium acetate, 10 mM Tris, pH 7.5, 60%ethanol) to remove impurities and salts. The membranes were vacuum driedfor 3 minutes on the vacuum manifold. The binding columns weretransferred to 50 ml collection tubes for elution. One ml ofnuclease-free water was applied to each membrane and incubated for 1minute at 23° C. The column assemblies were centrifuged, using aswinging bucket rotor, at 2,000×g for 2 minutes to collect the purifiedtotal RNA. The elution steps were repeated, with an additional 1 ml ofnuclease-free water. The second eluates were collected in fresh 50 mltubes. The purified total RNA samples were analyzed by spectrophotometryand agarose gel analysis. Total RNA yields were determined by absorbanceat 260 nm. The results are shown in Table 4 and FIGS. 4A and 4B. TABLE 4Total RNA Yield from 300 mg Rat Liver Total RNA Yield Total RNA YieldTotal RNA Yield Sample ID Elution 1 (μg) Elution 2 (μg) Elution 1 + 2(μg) 0.25 g Z3-1 1054.7 699.7 1754.4 0.25 g Z3-2 1141.2 652.9 1794.1 0.5g Z3-1 1017.2 626.9 1644.1 0.5 g Z3-2 987.7 551.5 1539.2 0.75 g Z3-1997.7 632.0 1629.7 0.75 g Z3-2 853.7 606.2 1459.9 1.0 g Z3-1 700.2 533.81233.8 1.0 g Z3-2 724.9 549.6 1274.5 1.5 g Z3-1 486.2 498.8 985.0 1.5 gZ3-2 728.5 469.0 1197.5 0.5 g Z3-1 S 1040.9 617.7 1658.6 0.5 g Z3-2 S1028.6 564.2 1592.8

FIG. 4A shows a gel analysis of purified total RNA from rat liver. RNAsamples were analyzed by electrophoresis on a native, 1% agarose, 1× TBEgel, stained with ethidium bromide. Ten μl of each sample was loaded perlane. The image was collected using an Alpha Innotech FluorChem™ ImagingSystem. Lanes 1-12 show the Elution 1 results and Lanes 13-24 show theElution 2 results. Lanes 1, 13: 0.25 g Z3-1; Lanes 2, 14: 0.25 g Z3-2;Lanes 3, 15: 0.5 g Z3-1; Lanes 4, 16: 0.5 g Z3-2; Lanes 5, 17: 0.75 gZ3-1; Lanes 6, 18: 0.75 g Z3-2; Lanes 7, 19: 11.0 g Z3-1; Lanes 8, 20:11.0 g Z3-2: Lanes 9, 21: 1.5 g Z3-1; Lanes 10, 22: 1.5 g Z3-2; Lanes11, 23: 0.5 g Z3-1 S; Lanes 12, 24: 0.5 g Z3-2 S. Lane M: Promega's PCRMarkers (Cat.# G3161), 1,000 bp, 750 bp, 500 bp, 300 bp, 150 bp and 50bp. Representative size markers are indicated. The undenatured 28Sribosomal RNA, 18S ribosomal RNA and small RNAs are indicated by arrows.

FIG. 4B show the results of a DNA contamination assay. Purified totalRNA samples were digested with Promega's RNase ONE™ Ribonuclease (Cat.#M4265). A 20 μl digestion mix, consisting of 4 μl of 10× ReactionBuffer, 14 μl of Nuclease-Free Water and 2 μl of RNase ONE™ Ribonuclease(5-10 u/μl) was mixed with 20 μl of each total RNA sample. Thedigestions were incubated at 37° C. for 1 hour and then held at 4° C.,until analyzed. The digests were analyzed by electrophoresis on anative, 1% agarose, 1× TBE gel, stained with ethidium bromide. Ten μl ofeach reaction was loaded per lane. The image was collected using anAlpha Innotech FluorChem™ Imaging System. Lanes 1-12 show the Elution 1results and Lanes 13-24 show the Elution 2 results. Lanes 1, 13: 0.25 gZ3-1; Lanes 2, 14: 0.25 g Z3-2; Lanes 3, 15: 0.5 g Z3-1; Lanes 4, 16:0.5 g Z3-2; Lanes 5, 17: 0.75 g Z3-1; Lanes 6, 18: 0.75 g Z3-2; Lanes 7,19: 11.0 g Z3-1; Lanes 8, 20: 11.0 g Z3-2: Lanes 9, 21: 1.5 g Z3-1;Lanes 10, 22: 1.5 g Z3-2; Lanes 11, 23: 0.5 g Z3-1 S; Lanes 12, 24: 0.5g Z3-2 S. Lane M: Promega's PCR Markers (Cat.# G3161), 1,000 bp, 750 bp,500 bp, 300 bp, 150 bp and 50 bp. Representative size markers areindicated. Partially degraded total RNA from the RNase ONE™ Ribonucleasedigestion is indicated in lanes 1-12 by an arrow.

Example 5 Isolation of Total RNA from Plant Cells

This example describes the isolation of total RNA from plant cells. 0.73g and 0.59 g canola leaves were flash frozen in liquid nitrogen andground to a fine powder with mortar and pestle, and then stored in 15 mltubes at −80° C. Two 15 ml tubes containing frozen ground canola leaveswere removed from the −80° C. freezer. 4 ml SV RNA Lysis Buffer (aguanidine thiocyanate (GTC) and β-mercaptoethanol (BME)-based solution;Promega cat# Z3051) including 80 ul of 48.7% beta mercaptoethanol wasadded to one tube containing ground canola leaves and the contents weretransferred into the second tube. An additional 4 ml of SV RNA LysisBuffer with BME was added to tube 1 and after mixing was transferred tothe second tube. Tube 1 was then discarded. The tube containing ˜1.3 gtissue and 8 ml SV RNA Lysis buffer with BME was incubated at 22° C. for20 minutes to lyse the tissue.

2 ml aliquots of the lysed canola tissue were dispensed into four 15 mltubes labeled P1, P2, P3 and P4. 4 ml DB4/zeolite mix was added to eachof the four tubes and they were inverted 3-4 times to mix the samples.DB4/zeolite mix was made by adding zeolite particles (Molecular Sievestype 13X, Sigma cat# M3010) to a Dilution Buffer at a concentration of125 mg/ml, where the dilution buffer was composed of 3M NaCl, 0.3M Na₃citrate•2H₂O, 0.2% SDS, and 0.0009% Blue dye (FD&C Blue #1), with thefinal pH adjusted to pH 4.0 with concentrated hydrochloric acid. TubesP1-P4 were incubated in a water bath at 70° C. for 3 minutes. The tubeswere returned to a rack at room temperature. An additional 500 mgzeolite powder was added to each of tubes P1 and P2 which were theninverted and shaken to mix.

For each sample a Promega PureYield™ Clearing Column was inserted in 50ml polypropylene catch tube (caps discarded) labeled P1-P4. One at atime, the 15 ml tubes P1-P4 were inverted and shaken to mix and thenimmediately poured into the PureYield™ Clearing Column in thecorresponding labeled tube. These clearing columns in catch tubes werecentrifuged at 2000×g for 10 minutes at 24° C. All the liquid passedthrough the columns. The Clearing Columns were discarded. 4 ml isopropylalcohol was added to the flow through in each tube and inverted 2-4times to mix. This mixture was poured into a Promega PureYield™ BindingColumn in a 50 ml tube and spun in a centrifuge at 2000×g at 24° C.

The binding columns were connected to a vacuum manifold (Promega cat#A7231). The columns were washed with 10 ml and then 20 ml SV RNA WashSolution (Promega cat #Z3091) applying a vacuum. After all wash solutionhad flowed through, the vacuum was applied for an additional 3 minutesto dry the column membrane. The binding columns were transferred toclean 50 ml tubes.

To elute the RNA, 1 ml of nuclease-free water was applied to the bindingmembrane and incubated at 22° C. for 1 minute. The columns were spun ina centrifuge at 2000×g for 2 minutes (elution 1). The column wastransferred to a fresh 50 ml tube and the elution with 1 mlnuclease-free water was repeated (elution 2).

To evaluate the eluates the samples were loaded on gels and on theAgilent 2100 Bioanalyzer. For the gel, 10 ul of each first elution (E1)was incubated at 37° C. for approximately one hour with 1.0 ul RNaseONE™ ribonuclease enzyme (Promega part # M426C) and 1.2 ul 10× RNaseONE™ buffer (Promega part #M217A) to digest the RNA. Approximately 3 ulof Blue/Orange 6× loading dye (Promega cat.# G1881) was added to theRNase ONE™-treated samples. 2 ul of Blue/Orange 6× loading dye was addedto 10 ul of untreated E1. Each lambda marker lane has 2 ul of lambda DNAEcoRI/HindIII marker (Promega Cat. # G173A) mixed with 8 ul water and 2ul Blue/Orange Loading Dye, 6×. Each 100 bp ladder lane has 3 ul 100 bpDNA ladder (Promega cat.# G20A) mixed with 7 ul water and 2 ulBlue/Orange Loading Dye, 6×. All samples with loading dye were loaded ona 1× TBE 1% agarose gel containing ethidium bromide. The gel shows thepresence of RNA for all four replicates and the ribonuclease-treatedsamples indicate that there is no visible DNA contamination.

The samples were also analyzed using the RNA 6000 Nano LabChip kit(Agilent Technologies, Palo Alto, Calif., USA, part# 5065-4476) with theRNA 6000 Ladder (Ambion, Austin, Tex., USA, Cat#7152) on the Agilent2100 Bioanalyzer. 1.0 ul of sample was loaded as recommended in theReagent Kit Guide RNA 6000 Nano Assay (Agilent Technologies, EditionNovember 2003). The Bioanalyzer electropherograms show sharp peaks thatindicate the RNA is intact and not degraded. They also show a return tobaseline between the two largest rRNA peaks (running at 39 and 44seconds), which is important as the inter-region between the two largestpeaks is typically where DNA contamination is seen if it is present.

Example 6 Removal of DNA Contamination from RNA Samples Isolated UsingTRIzol®

This example describes the removal of DNA contamination from RNA samplesoriginally isolated using TRIzol® reagent (Invitrogen Life Technologies,Carlsbad, Calif., cat#15596-026). Frozen bovine liver tissue washomogenized in TRIzol reagent in two tubes (A and B) with a rotor statorhomogenizer. Tube A had 1.21 g bovine liver and 12.1 ml TRIzol reagentwhile tube B had 1.15 g bovine liver and 11.5 ml TRIzol reagent. Thehomogenates in tubes A and B were combined, and inverted and shaken tomix. 3 ml of homogenate were dispensed into each of 6 Corex tubes. The 6tubes of homogenate were incubated at 22° C. for 5 minutes. 0.6 mlchloroform was added to each of the four tubes and then shakenvigorously by hand to mix for 15 seconds. The tubes were incubated at22° C. for 2-3 minutes then centrifuged at 10,700×g for 15 minutes at2-8° C. The colorless upper aqueous phase containing the RNA wastransferred to fresh 15 ml screw-cap polypropylene tubes.

The RNA was precipitated out of the aqueous solution by addition of 1.5ml isopropyl alcohol per tube and mixed. Immediately after isopropylalcohol was added, tubes 3 and 4 were each poured into PromegaPureYield™ Binding Columns in 50 ml tubes and the samples were preparedfollowing the Aqueous Control Protocol, see below. The remaining fourtubes were incubated at 22° C. for 40 minutes and then centrifuged at2600×g for 15 minutes at 2-8° C. The supernatant was removed. Tubes 5and 6 were prepared following the Pellet Hybrid Protocol, see below.Tubes 1 and 2 followed the TRIzol protocol (Invitrogen Form No. 18057N)with the pellets each being washed with 3 ml of 75% ethanol. Tubes 1 and2 were stored overnight at 4° C. in 75% ethanol.

Tubes 1 and 2 were mixed by vortex and then centrifuged at 2600×g for 5minutes at 2-8° C. The supernatants were removed and the RNA pellets airdried for 10 minutes at 22° C. Each of the RNA pellets was dissolved in1 ml nuclease-free water by pipetting up and down several times andincubating at 55-60° C. for 10 minutes. 50 ul of RNA solution wasremoved (called TRIzol RNA 1 and TRIzol RNA 2) for analysis beforeproceeding. 1 ml of SV RNA Lysis Buffer (a guanidine thiocyanate (GTC)and β-mercaptoethanol (BME)-based solution; Promega cat.# Z3051) with 20ul of 48.7% beta-mercaptoethanol was added to the remaining RNA (˜960ul) purified with TRIzol reagent. This ˜2 ml RNA/lysis buffer mixturewas used in place of the 2 ml lysate in the Promega PureYield™ RNA Midiprotocol, see below.

PureYield RNA Midi Protocol

4 ml DB4.36 (DB4.36 is 3M NaCl, 0.36M Na₃ citrate•2H₂O, 0.01 mM EDTA (pH8.0) and 0.0009% Blue dye (FD&C Blue #1) adjusted to a final pH of 4.0with concentrated hydrochloric acid) and 1 ml ZM1 (ZM1 is 2M NaCl, 0.1mM EDTA (pH 8.0) with zeolite added at a concentration of 0.45 g/ml)were added to each of tubes 1 and 2, which were then inverted 3-4 timeswhile shaking to mix. The tubes were incubated in a water bath at 70° C.for 3 minutes and then returned to a rack at 22° C. For each sample aPromega PureYield™ Clearing Column was inserted in a 50 ml polypropylenecatch tube (caps discarded). One at a time, the 15 ml tubes wereinverted and shaken to mix and then immediately poured into thePureYield™ Clearing Column in the corresponding labeled tube. Theseclearing columns in catch tubes were centrifuged at 2000×g for 10minutes at 24° C. The Clearing Columns were discarded. 4 ml isopropylalcohol was added to the flow through in each tube and inverted 2-4times to mix. This mixture was poured into a Promega PureYield™ BindingColumn in a 50 ml tube and centrifuged at 2000×g at 24° C. The BindingColumn flow through was discarded.

15 ml SV RNA Wash Solution (Promega cat #Z3091) was applied to theBinding Column and removed by centrifugation at 2,000×g for 5 minutes at24° C. The flow through was discarded. Another 15 ml SV RNA WashSolution was applied to the binding column and removed by centrifugationat 2,000×g for 10 minutes at 24° C. The Binding Columns were transferredinto fresh 50 ml tubes.

To elute the RNA, 1 ml of nuclease-free water was applied to the bindingmembrane and incubated at 22° C. for 1 minute. The columns werecentrifuged at 2000×g for 2 minutes at 24° C. These samples are calledTRIzol/PureYield 1 E1 and TRIzol/PureYield 2 E1. The elution of RNA wasrepeated with an additional 1 ml of nuclease-free water applied to thebinding membrane and the samples were called TRIzol/PureYield 1 E2 andTRIzol/PureYield 2 E2.

Aqueous Control Protocol (No Guanidine and No Zeolite)

This Aqueous Control protocol starts with the contents of tubes 3 and 4having been poured onto binding columns above, each containing anaqueous solution with RNA precipitating. The samples were centrifuged at2000×g for 10 minutes at 24° C. The binding column flow through wasdiscarded.

20 ml SV RNA Wash Solution (Promega cat #Z3091) was applied to theBinding Column and removed by centrifugation at 2,000×g for 10 minutesat 24° C. The flow through was discarded. Another 20 ml SV RNA WashSolution was applied to the binding column and removed by centrifugationat 2,000×g for 10 minutes at 24° C. The Binding Columns were transferredinto fresh 50 ml tubes. The binding columns were stored at −20° C. Thebinding columns were warmed at 4° C. and then at 22° C. The bindingcolumns were centrifuged at 2000×g for 5 minutes at 24° C. Then thebinding columns were transferred to a fresh 50 ml tube.

To elute the RNA, 1 ml of nuclease-free water was applied to the bindingmembrane and incubated at 22° C. for 1 minute. The columns werecentrifuged at 2000×g for 2 minutes at 24° C. These samples are called 3Aqueous Control E1 and 4 Aqueous Control E1. The elution of RNA wasrepeated with an additional 1 ml of nuclease-free water applied to thebinding membrane and the samples were called 3 Aqueous Control E2 and 4Aqueous Control E2.

Pellet Hybrid Protocol

This variation on the protocol starts with tubes 5 and 6 from above,each containing a TRIzol RNA pellet with the supernatant removed. Thepellets were dissolved by adding 2 ml SV RNA Lysis Buffer, containing 40ul 48.7% BME and stored overnight at 4° C.

Tubes 5 and 6 were returned to 22° C. and the buffer was pipetted up anddown to dissolve the RNA pellet. 4 ml DB4.36 and 1 ml ZM1 were added toeach tube, which were then inverted 3-4 times while shaking to mix. Thetubes were incubated in a water bath at 70° C. for 3 minutes and thenreturned to a rack at 22° C. For each sample a Promega PureYield™Clearing Column was inserted in a 50 ml polypropylene catch tube (capsdiscarded). One at a time, the 15 ml tubes were inverted and shaken tomix and then immediately poured into the PureYield™ Clearing Column inthe corresponding labeled tube. These clearing columns in catch tubeswere centrifuged at 2000×g for 10 minutes at 24° C. The Clearing Columnswere discarded. 4 ml isopropyl alcohol was added to the flow through ineach tube and inverted 2-4 times to mix. This mixture was poured into aPureYield™ Binding Column in a 50 ml tube and centrifuged at 2000×g at24° C. The Binding Column flow through was discarded.

15 ml SV RNA Wash Solution (Promega cat #Z3091) was applied to theBinding Column and removed by centrifugation at 2,000×g for 5 minutes at24° C. The flow through was discarded. Another 15 ml SV RNA WashSolution was applied to the binding column and removed by centrifugationat 2,000×g for 10 minutes at 24° C. The Binding Columns were transferredinto fresh 50 ml tubes.

To elute the RNA, 1 ml of nuclease-free water was applied to the bindingmembrane and incubated at 24° C. for 1 minute. The columns werecentrifuged at 2000×g for 2 minutes. These samples were called 5 PelletHybrid E1 and 6 Pellet Hybrid E1. The elution of RNA was repeated withan additional 1 ml of nuclease-free water applied to the bindingmembrane and the samples were called 5 Pellet Hybrid E2 and 6 PelletHybrid E2.

Analysis

After dilution in nuclease-free water, the nucleic acid yields weredetermined by absorbance at 260 nm (A260). TABLE 5 Total RNA Yield fromBovine Liver Cells A260 A260/ Vol total Avr. Sample Dilution A230 A260A280 280 (ul) (ug) of 2 TRIzol RNA 1 20 0.52638 1.11480 0.65915 1.69 900802.7 TRIzol RNA 2 40 0.26912 0.60010 0.37276 1.61 900 864.1 833.4TRIzol/PureYield 1 E1 20 0.28187 0.59267 0.29414 2.01 900 426.7TRIzol/PureYield 2 E1 20 0.31320 0.64764 0.31411 2.06 900 466.3 446.5 3Aqueous Control E1 20 0.40800 0.87175 0.42798 2.04 900 627.7 4 AqueousControl E1 20 0.37554 0.80448 0.39449 2.04 900 579.2 603.4 5 PelletHybrid E1 20 0.28248 0.57867 0.28290 2.05 900 462.9 6 Pellet Hybrid E120 0.25663 0.49269 0.24119 2.04 900 394.2 385.7 TRIzol/PureYield 1 E2 100.08574 0.13813 0.08249 1.67 900 49.7 TRIzol/PureYield 2 E2 10 0.094230.17587 0.09780 1.80 900 63.3 56.5 3 Aqueous Control E2 10 0.294940.59332 0.31480 1.88 900 213.6 4 Aqueous Control E2 10 0.30789 0.621190.32465 1.91 900 223.6 218.6 5 Pellet Hybrid E2 10 0.13116 0.252670.13164 1.92 900 91.0 6 Pellet Hybrid E2 10 0.13131 0.19240 0.10171 1.89900 69.3 80.1RNase ONE™ Ribonuclease Treatment

For each of the samples listed above, 10 ul of sample was added to 1.2ul of RNase ONE 10× Buffer (Promega Part #217A) in a PCR strip tube andthen 1.0 ul of RNase ONE Ribonuclease enzyme (Promega part # M4261) wasadded. The strip tube was capped and incubated at 37° C. for about anhour.

Agarose Gel

2.5 ul of Blue/Orange Loading Dye, 6× (Promega cat.# G1881) was added toeach ribonuclease-treated sample. On a piece of parafilm, 10 ul ofuntreated sample was added to 2 ul of Blue/Orange Loading Dye, 6×. Eachlambda marker lane has 2 ul of lambda DNA EcoRI/HindIII marker (Promegacat.# G173A) mixed with 8 ul water and 2 ul Blue/Orange Loading Dye, 6×.Each 100 bp ladder lane has 3 ul 100 bp DNA ladder (Promega cat. #G6951)mixed with 7 ul water and 2 ul Blue/Orange Loading Dye, 6×.

The samples were electrophoresed on a 1% agarose 1× TBE gel withethidium bromide added. The results are shown in FIG. 5. FIG. 5A showsthe untreated samples and FIG. 5B shows the samples treated with RNaseONE™ Ribonuclease. Both FIGS. 5A and 5B have samples loaded in the sameorder, with the following lane designations: lane 1—Lambda DNAEcoRI/HindIII marker; lane 2-100 bp DNA ladder (FIG. 5A only); lane3—blank; lane 4—TRIzol RNA 1 E1; lane 5—TRIzol RNA 2 E1; lane6—TRIzol/PureYield 1 μl; lane 7—TRIzol/PureYield 2 μl; lane 8-3 AqueousControl E1; lane 9-4 Aqueous Control E1; lane 10-5 Pellet Hybrid E1;lane 11-6 Pellet Hybrid E1; lane 12-blank; lane 13—Lambda DNAEcoRI/HindIII marker; lane 14-100 bp DNA Ladder; lane 15—blank; lane16—TRIzol/PureYield 1 E2; lane 17—TRIzol/PureYield 2 E2; lane 18-3Aqueous Control E2; lane 19-4 Aqueous Control E2; lane 20-5 PelletHybrid E2; lane 21-6 Pellet Hybrid E2; lane 22—blank; lane 23—Lambda DNAEcoRI/HindIII marker; lane 24-100 bp DNA Ladder (FIG. 5A only).

The results presented in FIG. 5, particularly FIG. 5B, show that theprotocols using only TRIzol reagent had detectable DNA contaminationwhile the protocols which added DB4 and zeolite to the TRIzol reagentprotocols resulted in no detectable DNA contamination. In FIG. 5B, lanes4, and 5 (from the first elution) and lanes 18 and 19 (from the secondelution) show the result of the DNA contamination assay where onlyTRIzol reagent is used to purify the RNA, while lanes 8 and 9 show theresults of the DNA contamination assay where TRIzol reagent was usedfollowed by washes on a PureYield binding column. As seen in thisfigure, all of these lanes display visible DNA contamination. Lanes 6,7, 10, and 11 (from the first elution) and lanes 16, 17, 20 and 21 (fromthe second elution) show the result of the DNA contamination assay whereDB4 (dilution buffer at pH 4) and zeolite particles are used to removeDNA. As seen in this figure, not detectable DNA contamination is presentin these lanes. Together, these results indicate that a protocolemploying DB4 and zeolite particles is able to purify RNA away from DNAat a level that is superior to the level achieved with a protocolemploying only TRIzol reagent.

Example 7 Isolation of Total RNA from Human Blood

This example describes the isolation of total RNA from human blood.Blood drawn from two individuals was designated “(H)” for “High” and“(L)” for “Low” to indicate the relative white blood cell (leukocyte)count. The blood was collected by venipuncture in sterile 6 ml K₂EDTA BDVacutainer® Plus Plastic Blood Collection Tubes (Becton Dickinson,Franklin Lakes, N.J.). The blood was stored at 4° C. for 4 days and thentransferred to ice for processing. Four tubes of blood (estimated tocontain approximately 5 ml each) from each individual were pooled anddispensed into 1.5 ml aliquots in 2 ml microcentrifuge tubes with sealedcaps. The (H) samples were centrifuged at 12,000×g for 15 minutes at 4°C. and the (L) samples were centrifuged at 9,000×g for approximately 10minutes at 4° C. to fractionate the blood. The upper, yellow, clearserum layer was carefully removed with a pipet. The white buffy coat,containing leukocytes, was carefully withdrawn from the interface with apipet. The buffy coat was pooled for each individual. The pooled buffycoat samples were contaminated with some red blood cells from the lowerred blood cell fraction. Two ml of Lysis Buffer (4M guanidinethiocyanate, 10 mM Tris, pH 7.5, with beta-mercaptoethanol addedseparately to a final concentration of 0.974% (v/v)) was added to eachof the samples, (H) and (L). The mixtures were vortexed vigorously tolyse the leukocytes. The dark red lysates were stored at −70° C., untiluse.

The frozen lysates were thawed at 4° C. Four ml of Dilution Buffer (3MNaCl, 0.3M Na₃ citrate•2H₂O, 0.2% SDS, 0.0009% Blue dye (FD&C Blue #1),adjusted to a final pH of 4.0 with concentrated hydrochloric acid (HCl))was added to each sample and mixed by inversion. The mixtures congealed,forming insoluble, stringy clumps. The tubes were incubated at 70° C. ina hybridization oven for 3 minutes and then placed at ambienttemperature (23° C.). Molecular Sieves, Type 13X, powder (<10 μm)(zeolite) was weighed and 0.5 g was added to each tube. Each mixture wasshaken vigorously and immediately poured into a Promega PureYield™clearing column, nested in a 50 ml collection tube. The columns werecentrifuged in a swinging bucket rotor at approximately 2,000×g, 23° C.for 10 minutes. The cleared lysates, containing RNA, were captured in 50ml collection tubes.

The following steps were performed at ambient temperature (23° C.). Fourml of isopropanol was added to each of the cleared lysates and mixed byinversion. Each mixture was applied to a Promega PureYield™ bindingcolumn, attached to a vacuum manifold. A vacuum of approximately 15 in.Hg was applied to the columns. Each sample passed through the column,leaving the RNA bound to the binding column membrane. The membranes werewashed twice with 10 ml and then 20 ml of Wash Solution (60 mM potassiumacetate, 10 mM Tris, pH 7.5, 60% ethanol) to remove impurities andsalts. The membranes were vacuum dried for 3 minutes on the vacuummanifold. The binding columns were transferred to 50 ml collection tubesfor elution.

0.5 ml of nuclease-free water was applied to each membrane and incubatedfor 1 minute at 23° C. The column assemblies were centrifuged, using aswinging bucket rotor, at 2,000×g for 2 minutes to collect the purifiedtotal RNA. The elution steps were repeated, with an additional 0.5 ml ofnuclease-free water. The second eluates were collected in fresh 50 mltubes. Approximately 200 μl and 500 μl of eluate was recovered from thefirst and second elutions, respectively. The reduced eluate volume wascaused by absorption of water by the dried binding membrane. Thepurified total RNA samples were analyzed by spectrophotometry andagarose gel analysis. Total RNA yields were determined by absorbance at260 nm. The results are shown in Table 6 and FIGS. 6A and 6B. TABLE 6Total RNA Yield from Human Blood Total RNA Yield Total RNA Yield TotalRNA Yield Sample ID Elution 1 (μg) Elution 2 (μg) Elution 1 + 2 (μg) Hu(H) 12.1 29.2 41.3 Hu (L) 9.1 8.2 17.3

FIG. 6A shows a gel analysis of purified total RNA from human blood. RNAsamples were analyzed by electrophoresis on a native, 1% agarose, 1× TBEgel, stained with ethidium bromide. Ten μl of each sample was loaded perlane. The image was collected using an Alpha Innotech FluorChem™ ImagingSystem. The image brightness was increased to visualize the faint rRNAbands. Lanes 1-2 show the Elution 1 results and Lanes 3-4 show theElution 2 results. The Elution 1 samples were more concentrated than theElution 2 samples. Lanes 1, 3: Hu (H); Lanes 2, 4: Hu (L). Theundenatured 28S ribosomal RNA, 18S ribosomal RNA and small RNAs areindicated by arrows.

FIG. 6B shows the results of a DNA contamination assay of total RNApurified from human blood. Purified total RNA samples were digested withPromega's RNase ONE™ Ribonuclease (Cat.# M4265). A 15 μl digestion mix,consisting of 3 μl of 10× Reaction Buffer, 9 μl of Nuclease-Free Waterand 3 μl of RNase ONE™ Ribonuclease (5-10 u/μl) was mixed with 15 μl ofeach total RNA sample. The digestions were incubated at 37° C. for 1hour and then held at 4° C., until analyzed. The digests were analyzedby electrophoresis on a native, 1% agarose, 1× TBE gel, stained withethidium bromide. Ten μl of each reaction was loaded per lane. The imagewas collected using an Alpha Innotech FluorChem™ Imaging System. Lanes1-2 show the Elution 1 results and Lanes 3-4 show the Elution 2 results.Lanes 1, 3: Hu (H); Lanes 2, 4: Hu (L). Lane G: Control samplescontaining genomic DNA.

Example 8 DNA Zeolite Binding

This example describes assays employed to determine the amount of DNAand RNA binding to zeolite particles in the presence of an acidicbuffer. As described below, zeolite particles preferentially bind to DNAmolecules and have only limited binding to RNA molecules.

7.2 g frozen bovine spleen tissue was homogenized in 48 ml SV RNA LysisBuffer (Promega cat# Z3051) including 960 ul of 48.7% betamercaptoethanol (BME, Promega part#Z523A) using a rotor statorhomogenizer. The homogenate was divided into tubes and stored at minus80° C. All the tubes used in this example were polypropylene screw-capcentrifuge tubes.

A tube containing homogenized bovine spleen was removed from the −80° C.freezer and thawed in water at 4° C. 2 ml aliquots of the bovine spleenhomogenate were dispensed into four 15 ml tubes and the remainder of thehomogenate was stored at −20° C. Four (4) ml DB4.36 was added to each ofthe tubes and then 1 ml ZM1 was added to each tube. DB4.36 is 3M NaCl,0.36M Na₃ citrate•2H₂O, 0.1 mM EDTA (pH 8.0), and 0.0009% Blue dye (FD&CBlue #1) adjusted to a final pH of 4.0 with concentrated hydrochloricacid, while ZM1 is 2M NaCl, 0.1 mM EDTA (pH 8.0) with zeolite added at aconcentration of 0.45 g/ml. The tubes were inverted 3-4 times and shakento mix the samples. The 4 tubes were incubated in a water bath at 70° C.for 3 minutes and then returned to a rack at room temperature.

For each sample a PureYield Clearing Column (Promega) was inserted in a50 ml polypropylene catch tube (cap discarded) labeled C1-C4. One at atime, the 15 ml tubes 1-4 were inverted and shaken to mix and thenimmediately poured into the PureYield Clearing Column in thecorresponding labeled tube. These clearing columns in catch tubes werecentrifuged at 2000×g for 10 minutes at 22° C. The Clearing Columns weresaved to later separate the zeolite from the clearing column membrane,see below. 4 ml isopropyl alcohol was added to the flow through in eachtube and inverted 2-4 times to mix. This mixture was poured into aPureYield Binding Column (Promega), labeled B1-B4, in a 50 ml tube andspun in a centrifuge at 2000×g at 22° C.

The binding columns were transferred to a fresh 50 ml column and theflow through (FT 1-4) was stored at −20° C. 20 ml SV RNA Wash Solution(Promega cat #Z3091) was added to the binding column and centrifuged at2,000×g for 5 minutes. The flow through was stored at −20° C. Then 10 mlSV RNA Wash Solution was added to the binding column and centrifuged at2,000×g for 10 minutes. The binding columns were transferred to clean 50ml tubes.

To elute the RNA, 1 ml of nuclease-free water was applied to the bindingmembrane and incubated at 22° C. for 1 minute. The columns werecentrifuged at 2000×g for 2 minutes (RNA elution 1). The column wastransferred to a fresh 50 ml tube and the elution with 1 mlnuclease-free water was repeated (RNA elution 2).

The used zeolite in clearing columns C1-C4 was removed from the columnmembrane by separating the zeolite from the membrane and then scrapingoff any remaining membrane. The zeolite was transferred to a freshPureYield Clearing Column labeled C1′-C4′ in a 50 ml tube. 1.0 mlnuclease-free water was added to each clearing column C1′-C4′ andincubated at 22° C. for 3 minutes. The tubes were then centrifuged at2,000×g for 10 minutes at 22° C. (zeolite elution 1). The clearingcolumns were transferred to fresh 50 ml tubes and the elution with 1 mlnuclease-free water was repeated (zeolite elution 2).

The first sample analysis was performed as follows to generate FIG. 7 a.The samples were loaded on 1% Seakem Gold, 1× TBE gels containingethidium bromide (Cambrex Bio Science Rockland, Inc., Rockland, Me.Cat#54907). 15 ul of zeolite (1-4) elution 1 was added to 3 ulBlue/Orange Loading Dye, 6× (Promega). 15 ul of flow through sample(FT1-4) was added to 3 ul Blue/Orange Loading Dye, 6×. 15 ul ofhomogenate was added to 3 ul Blue/Orange Loading Dye, 6×. Each lambdamarker lane has 1 ul of lambda DNA EcoRI/HindIII marker (Promega) mixedwith 9 ul water and 2 ul Blue/Orange Loading Dye, 6×. The zeoliteelutions in lanes 2-5 show that certain nucleic acids are bound andreleased from the zeolite. In FIG. 7 a, the sample loading order is asfollows: 1. Lambda DNA EcoRI/HindIII marker; 2. Zeolite 1 elution 1; 3.Zeolite 2 elution 1; 4. Zeolite 3 elution 1; 5. Zeolite 4 elution 1; 6.Lambda DNA EcoRI/HindIII marker; 7. Blank; 8. Flow through 1; 9. Blank;10. Flow through 2; 11. Blank; 12. Flow through 3; 13. Blank; 14. Flowthrough 4; 15. Blank; 16. Lambda DNA EcoRI/HindIII marker; 17. Blank;18. Homogenate; 19. Blank; and 20. Blank. The gel in FIG. 7 a shows theenormous amount of DNA and RNA in the homogenate in lane 18, and thatvery little nucleic acid remains in the PureYield Binding Column flowthrough in lanes 8, 10, 12 and 14.

The second sample analysis was performed as follows to generate FIG. 7b. 100 ul of each zeolite (1-4) elution 1 sample was incubated at 37° C.for two hours with 3.0 ul RNase ONE™ ribonuclease enzyme (Promega part #M426C) and 11 ul 10× RNase ONE™ buffer (Promega part# M217A) to digestthe RNA. 100 ul of each zeolite (1-4) elution 1 sample was incubated at37° C. for two hour with 3.0 ul RQ1 RNase-free DNase (Promega) and 11 ul10× Reaction Buffer (Promega) to digest the DNA. After the incubationthe samples were pipetted onto parafilm and concentrated by evaporationfor one hour. 25 ul of concentrated sample was mixed with 5 ulBlue/Orange Loading Dye, 6× (Promega) and 20 ul was loaded on the gel.Each lambda marker lane has 2 ul of lambda DNA EcoRI/HindIII marker(Promega) mixed with 8 ul water and 2 ul Blue/Orange Loading Dye, 6×.Each 100 bp ladder lane has 3 ul 100 bp DNA ladder (Promega) mixed with7 ul water and 2 ul Blue/Orange Loading Dye, 6×. All samples withloading dye were loaded on a 1×TBE, 1% agarose LE analytical grade(Promega) gel containing ethidium bromide. The RNase treatment and DNasetreatment show that both RNA and DNA are bound and eluted from thezeolite. However, it is clear that zeolite only binds a small amount ofRNA and is preferentially binding to DNA. Moreover, it appears that thezeolite binds the DNA strongly such that, using the conditions describedabove, not much of the DNA was eluted from the zeolite particles (i.e.most of the DNA remained bound to the zeolite particles).

In FIG. 7 b, the sample loading order is as follows: 1. 100 bp DNAladder; 2. Lambda DNA EcoRI/HindIII marker; 3. Blank; 4. Zeolite 1elution 1; 5. Blank; 6. Blank 7. Zeolite 2 elution 1; 8. Blank; 9.Blank; 10. Zeolite 3 elution 1; 11. Blank; 12. Blank; 13. Zeolite 4elution 1; 14. Blank; 15. Blank; 16. Lambda DNA EcoRI/HindIII marker;17. Blank; 18. Zeolite 1 elution 1, treated with DNase; 19. Blank; 20.Zeolite 2 elution 1, treated with DNase; 21. Blank; 22. Zeolite 3elution 1, treated with DNase; 23. Blank; 24. Zeolite 4 elution 1,treated with DNase; 25. Blank; 26. Lambda DNA EcoRI/HindIII marker; 27.Blank; 28. Zeolite 1 elution 1, treated with RNase; 29. Blank; 30.Zeolite 2 elution 1, treated with RNase; 31. Blank; 32. Zeolite 3elution 1, treated with RNase; 33. Blank; 34. Zeolite 4 elution 1,treated with RNase; 35. Blank; 36. Lambda DNA EcoRI/HindIII marker; 37.100 bp DNA ladder. The DNase-treated samples in lanes 18, 20, 22 and 24show distinct RNA bands as well as a smear. The RNase-treated samplesshow a faint smear of DNA in lanes 28, 30, 32, and 34.

The third sample analysis was performed as follows to generate FIG. 7 c.10 ul of each RNA elution 1 sample (#1, 2, and 4) was incubated at 37°C. for two hours with 2.0 ul RNase ONE™ ribonuclease enzyme (Promega)and 1.3 ul 10× RNase ONE™ buffer (Promega) to digest the RNA. RNA 3elution 1 was incubated at the same temperature and for the same time asthe other samples, but had 4.0 ul RNase ONE ribonuclease enzyme with the1.3 ul 10× RNase ONE buffer. 3 ul Blue/Orange Loading Dye, 6× was addedto each RNase-treated RNA elution sample. 2 ul Blue/Orange Loading Dye,6× was added to 10 ul of each RNA elution sample. Each lambda markerlane has 2 μl of lambda DNA EcoRI/HindIII marker (Promega) mixed with 8ul water and 2 ul Blue/Orange Loading Dye, 6×. Each 100 bp ladder lanehas 2 ul 100 bp DNA ladder (Promega) mixed with 8 ul water and 2 μlBlue/Orange Loading Dye, 6×.

In FIG. 7 c, the sample loading order is as follows: 1. Lambda DNAEcoRI/HindIII marker; 2. Blank; 3. RNA 1 elution 1; 4. RNA 2 elution 1;5. RNA 3 elution 1; 6. RNA 4 elution 1; 7. Blank; 8. Lambda DNAEcoRI/HindIII marker; 9. 100 bp DNA ladder; 10. Blank; 11. RNA 1 elution1, treated with RNase; 12. RNA 2 elution 1, treated with RNase; 13. RNA3 elution 1, treated with RNase; 14. RNA 4 elution 1, treated withRNase; 15. Blank; and 16. Lambda DNA EcoRI/HindIII marker. The gel inFIG. 7C shows the RNA eluted from the PureYield Binding membrane inlanes 3-6. The RNase-treated samples in lanes 11-14 show little or noDNA eluted from the PureYield Binding membrane.

Example 9 Preferential DNA or RNA Binding with Various Substrates in anAcidic Buffer

This Example describes protocols used to assay various substrates fornucleic acid binding in the presence of an acidic buffer. Besidesdemonstrating that various zeolite particle solutions preferentiallybind DNA, these assays demonstrated that titanium oxide preferentiallybinds RNA.

600 mg frozen bovine spleen was homogenized per 2 ml SV RNA Lysis Buffer(Promega) including 20 ul 48.7% beta mercaptoethanol (BME, Promega) permilliliter buffer using a rotor stator homogenizer. The homogenate wasdivided into tubes and stored at minus 80° C.

Tubes containing homogenized bovine spleen were removed from the −80° C.freezer and thawed in water at 4° C. The homogenate was diluted with anequal amount of SV RNA Lysis Buffer with BME added. 2 ml aliquots of thediluted bovine spleen homogenate were dispensed into 15 ml tubes. 4 mlDB4 was added to each of the tubes that later had added zeolite powder,zeolite in solution or the non-zeolite binding matrices. DB4 is 3.0MNaCl, 0.30M Na₃ citrate•2H₂O, and 0.0009% Blue dye (FD&C Blue #1)adjusted to a final pH of 4.0 with concentrated hydrochloric acid. 4 mlreduced salt DB4 was added to a second set of tubes with zeolite powderor zeolite in solution. Reduced salt DB4 is 2.2M NaCl, 0.30M Na₃citrate•2H₂O, adjusted to a final pH of 4.0 with concentratedhydrochloric acid. After dilution buffer was added the tubes were mixedby inversion. The tubes were incubated in a water bath at 70° C. for 3minutes and then returned to a rack at room temperature. Then to each ofthe tubes, one of the following binding matrices was added: 0.5 gzeolite powder (Molecular Sieves, type 13X), 0.5 g zeolite in 1-2 mlsalt water (2-5M NaCl), 0.5 g zeolite in 1.0 ml water, 0.5 g zeolite in1.0 ml isopropanol, 0.5 g alumina in 1.0 ml water, 0.5 g Sea Sand, 0.5 gtitanium (IV) oxide anatase powder in 1.0 ml water, 0.25 g zinc oxide in1.0 ml water, 0.5 g zirconium oxide in 1.0 ml water. The tubes wereinverted and shaken to mix the samples.

For each sample a PureYield Clearing Column (Promega) was inserted in 50ml polypropylene catch tube (cap discarded). One at a time, the 15 mltubes were inverted and shaken to mix and then immediately poured into aPureYield Clearing Column. These clearing columns in catch tubes werecentrifuged at 2000×g for 10 minutes at 24° C. The clearing columns werediscarded. 4 ml isopropyl alcohol was added to the flow through in eachtube and inverted 2-4 times to mix. This mixture was poured into aPureYield Binding Column (Promega) in a 50 ml tube and spun in acentrifuge at 2000×g at 24° C.

The binding columns were transferred to a clean 50 ml tube. 20 ml SV RNAWash Solution (Promega, prepared following instructions) was applied tothe column and centrifuged at 2,000×g for 6 minutes. A second wash of 10ml SV RNA Wash Solution was added with an additional centrifugation at2,000×g for 6 minutes. The binding columns were transferred to clean 50ml tubes.

To elute the RNA, 1 ml of nuclease-free water was applied to the bindingmembrane and incubated at 22° C. for 1 minute. The columns were spun ina centrifuge at 2000×g for 2 minutes (elution 1). The column wastransferred to a fresh 50 ml tube and the elution with 1 mlnuclease-free water was repeated (elution 2).

FIG. 8 was generated as follows. 10 ul of each sample was incubated at37° C. for two hours with 1.0 ul RNase ONE™ ribonuclease enzyme (Promegapart # M426C) and 1.2 ul 10× RNase ONE™ buffer (Promega part# M217A) todigest the RNA. After the incubation the RNase treated samples were eachmixed with 2.5 ul Blue/Orange Loading Dye, 6× (Promega part#G190A) and10 ul of untreated sample mixed with 2.0 ul Blue/Orange Loading Dye, 6×.Each lambda marker lane has 2 ul of lambda DNA EcoRI/HindIII marker(Promega part# G173A) mixed with 8 ul water and 2 ul Blue/Orange LoadingDye, 6×. Each 100 bp ladder lane has 3 ul 100 bp DNA ladder (Promegapart# G20A) mixed with 7 μl water and 2 ul Blue/Orange Loading Dye, 6×.All samples with loading dye were loaded on a 1× TBE, 1% agarose LEanalytical grade (Promega) gel containing ethidium bromide.

In FIG. 8, the sample loading order is as follows: 1. Lambda DNAEcoRI/HindIII marker; 2. Zeolite powder A; 3. Zeolite powder B; 4.zeolite in 2M NaCl; 5. zeolite in water A; 6. zeolite in water B; 7.Zeolite in isopropanol; 8. zeolite powder with lower salt DB4; 9.zeolite in water with lower salt DB4; 10. zeolite in 5M NaCl with lowersalt DB4; 11. zeolite in 2.5M NaCl with lower salt DB4; 12. alumina; 13.sea sand; 14. titanium oxide; 15. zinc oxide; 16. zirconium oxide; 17.100 bp DNA ladder; 18. Lambda DNA EcoRI/HindIII marker; 19. Zeolitepowder A, treated with RNase; 20. Zeolite powder B, treated with RNase;21. zeolite in 2M NaCl, treated with RNase; 22. zeolite in water A,treated with RNase; 23. zeolite in water B, treated with RNase; 24.Zeolite in isopropanol, treated with RNase; 25. zeolite powder withlower salt DB4, treated with RNase; 26. zeolite in water with lower saltDB4, treated with RNase; 27. zeolite in 5M NaCl with lower salt DB4,treated with RNase; 28. zeolite in 2.5M NaCl with lower salt DB4,treated with RNase; 29. alumina, treated with RNase; 30. sea sand,treated with RNase; 31. titanium oxide, treated with RNase; 32. zincoxide, treated with RNase; 33. zirconium oxide, treated with RNase; and34. Lambda DNA EcoRI/HindIII marker.

The gel in FIG. 8 shows that titanium oxide binds RNA preferentially asshown by the DNA and not RNA present in the binding column elution(untreated sample in lane 14, RNase treated sample in lane 31.) Incontrast to the titanium oxide, the gel shows that zeolite in powderform or dissolved in water solutions binds most or all of the DNApresent in the lysate and leaves the RNA for elution from the bindingcolumn. The binding matrices alumina, sea sand, zinc oxide and zirconiumoxide show binding of some RNA as indicated by the loss or reduction inintensity of the smallest RNA band and reduction in band intensity forthe larger 18S and 28S rRNA bands.

Example 10 Zeolite Membranes, Silica-Zeolite Membranes andZeolite-Silica Particle Composites

This example describes generating zeolite membranes, silica-zeolitecoated membranes and zeolite-silica particle composites.

Zeolite membranes were generated as follows. 0.5 gm of zeolite (Aldrich,cat #233641, Molecular Sieves 3A, with a formula ofK_(n)Na_(12-n)[(AlO₂)₁₂(SiO₂)₁₂].xH₂O) was added to each of 2 Promegaclearing columns (cat# A2492) containing cellulose membranes, labeled as1A and 1B. 0.5 gm of zeolite (Aldrich cat #233641) was added to each of2 Promega binding columns containing silica membranes, labeled as 2A and2B. 0.5 gm of zeolite (Sigma-Aldrich cat #283592, Molecular sieves, 13×,with a formula of: Na₈₆[(AlO₂)₈₆(SiO₂)₁₀₆].xH₂O) was added to each of 2Promega binding columns containing silica membranes, labeled as 3A and3B. All 6 columns were inserted into 50 ml plastic tubes.

To each of the above columns, 1 ml of 93% (wt/vol) KOH was added, andthe contents mixed using a plastic pipette tip to thoroughly wet thezeolite particles. Columns were centrifuged at 2000×g for 5 minutes in aswinging bucket rotor. 2 ml of 1N HCl was then added and thetubes/columns were centrifuged at 2000×g for 5 minutes. A second 2 ml of1N HCl was then added (except for tube 6 in which 2 ml of nuclease-freewater was added) and the tubes/columns were centrifuged at 2000×g for 5minutes. 5 ml of nuclease free water (Promega) was then added and thetubes/columns were centrifuged at 2000×g for 5 minutes. A second 5 ml ofnuclease free water (Promega) was then added and the tubes/columns werecentrifuged at 2000×g for 5 minutes. All columns were transferred toclean 50 ml tubes for use in the following Example showing their utilityin removing DNA from RNA preparations.

Silica-zeolite composites were generated using Scotchlite™ S60 Glassbubbles (3M, St. Paul, Minn.). 0.6 gm of Scotchlite S60 glass bubbleswere added to a 50 ml plastic tube containing 1.3 gm of zeolite(Sigma-Aldrich cat #283592, Molecular sieves, 13×) and thoroughly mixed.4 ml of 93% KOH was added per tube and thoroughly mixed, then pouredinto a Promega clearing column inserted into a 50 ml tube. The tube wascentrifuged at 2000×g for 10 minutes in a swinging bucket rotor. Then 4ml of 1N HCl was added to the column, and the tube was centrifuged at2000×g for 10 minutes. Then a second 4 ml of 1N HCl was added to thecolumn, and the tube was centrifuged at 2000×g for 10 minutes. Then 5 mlof nuclease free water was added to the column, and the tube wascentrifuged at 2000×g for 10 minutes. Then a second 5 ml of nucleasefree water was added to the column, and the tube was centrifuged at2000×g for 10 minutes. The flow-through fluid in the tube was discarded.Then a third 5 ml of nuclease free water was added to the column, andthe tube was centrifuged at 2000×g for 10 minutes. The column contentswere transferred to a clean 50 ml plastic tube, and nuclease free waterwas added to 25 ml total volume.

Example 11 Using Zeolite Membranes and Silica-Zeolite Composites

This Example shows that zeolite membrane, silica-zeolite compositemembrane, and silica-zeolite composite in solution can remove DNA fromRNA in tissue sample purifications. 1.61 g and 1.69 g frozen bovineliver were homogenized in 21.5 ml and 22.5 ml SV RNA Lysis Buffer(Promega), respectively, including 430 ul and 450 ul of 48.7% betamercaptoethanol (BME, Promega) using a rotor stator homogenizer. The twohomogenates were mixed together. All the tubes used in this example werepolypropylene screw-cap centrifuge tubes.

2 ml aliquots of the bovine liver homogenate (lysate) were dispensedinto 15 ml tubes and the remainder of the lysate was stored at −80° C.Four (4.0) ml DB4.36 was added to each of the tubes with lysate. DB4.36is 3M NaCl, 0.36M Na₃ citrate•2H₂O, 0.1 mM EDTA (pH 8.0), and 0.0009%Blue dye (FD&C Blue #1) adjusted to a final pH of 4.0 with concentratedhydrochloric acid. The tubes were inverted 3-4 times and shaken to mixthe samples. The tubes were incubated in a water bath at 70° C. for 5minutes and then returned to a rack at room temperature. 0.5 ml S60Silica-zeolite composite was added to each of two tubes (containinglysate and dilution buffer) and heated for 5 minutes at 70° C. and thenreturned to a rack at room temperature. After cooling these two S60mixtures were shaken by inversion and transferred to a PureYield™ (PY)Binding column in tubes 4A and 4B. The remaining lysate/dilution buffermixtures were inverted and shaken to mix one at a time and immediatelytransferred to columns in tubes 1A, 1B, 2A, 2B, 3A, 3B, 5A, 5B, 6, 7, 8Aand 8B. The columns were swirled to mix. The columns in tubes 1A, 1B,2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 6 and 7 contain a membrane or zeolitepowder as described in Table 7. The negative control tubes 8A and 8Bhave only PureYield™ binding columns without any added zeolite orsilica. Tubes 1A and 1B were allowed to drip by gravity and the flowthrough was reapplied to the column 2 times. A small amount of flowthrough for tube 1A was lost in transfer. TABLE 7 Column type for Tube #clearing step 1A, 1B zeolite membrane (Aldrich, Type 3A) PY ClearingColumn 2A, 2B silica-zeolite membrane composite PY Binding Column batch2 (Aldrich, Type 3A) 3A, 3B silica-zeolite membrane composite PY BindingColumn batch 1 (Sigma, Type 13X cat#283592-1 KG) 4A, 4B S60Silica-zeolite composite in solution PY Binding Column 5A, 5B zeolitepowder (Aldrich Type 3A) PY Binding Column 6 KOH 1 Water membrane(Sigma, PY Binding Column Type 13X cat#283592-1 KG) 7 KOH 2 HCl membrane(Sigma, PY Binding Column Type 13X cat#283592-1 KG) 8A, 8B NegativeControl (no additional zeolite PY Binding Column or silica)

All tubes were centrifuged at 2000×g for 10 minutes at 24° C. Tubes 3Aand 3B were centrifuged for an additional S minutes at 2,000×g at 24° C.Tubes 2A, 2B, 8A and 8B were centrifuged for an additional 2 times at2000×g for S minutes. Negative control tube 8A had yet anothercentrifugation at 3000×g for 5 minutes at 24° C., but remained cloggedso was not processed beyond this step. 4 ml isopropyl alcohol was addedto the flow through in each tube and inverted and shaken well to mix.This mixture was poured into a correspondingly labeled PureYield™Binding Column (Promega) attached to a vacuum manifold and vacuum wasapplied. Negative control tube 8B as well as tubes 6 and 7 clogged andso were not processed beyond this step.

After the mixture passed through the column, 20 ml and then 10 ml SV RNAWash solution was added to the binding column with the vacuum appliedeach time until there was no standing wash solution on the columnmembrane. The vacuum was applied for an additional 3 minutes to dry themembrane. The binding columns were transferred to clean 50 ml tubes. Toelute the RNA, 1 ml of nuclease-free water was applied to the bindingmembrane and incubated at 22° C. for 2 minutes. The columns werecentrifuged at 2000×g for 3 minutes at 24° C.

FIGS. 9A and 9B were generated as follows. 10 ul of each sample wasincubated at 37° C. for two hours with 2.0 ul RNase ONE™ ribonucleaseenzyme (Promega) and 1.3 ul 10× RNase ONE™ buffer (Promega) to digestthe RNA. After the incubation the RNase treated samples were each mixedwith 2.5 ul Blue/Orange Loading Dye, 6× (Promega) and 10 ul of untreatedsample mixed with 2.0 ul Blue/Orange Loading Dye, 6×. Each lambda markerlane had 2 ul of lambda DNA EcoRI/HindIII marker (Promega) mixed with 8ul water and 2 ul Blue/Orange Loading Dye, 6×. Each 100 bp ladder lanehad 2 ul 100 bp DNA ladder (Promega) mixed with 8 ul water and 2 ulBlue/Orange Loading Dye, 6×. All samples with loading dye were loaded ona 1× TBE, 1% agarose gel containing ethidium bromide.

The samples were loaded in the same order for FIGS. 9A and 9B, the onlydifference was that 9B samples were treated with RNase ONE™ ribonucleasewhile the 9A samples were untreated. The sample loading order is asfollows: 1. Lambda DNA EcoRI/HindIII marker; 2. Blank; 3. Sample 1A,zeolite membrane; 4. Sample 1B, zeolite membrane; 5. Sample 2A,silica-zeolite membrane (Type 3A); 6. Sample 2B, silica-zeolite membrane(Type 3A); 7. Sample 3A, silica-zeolite membrane (Type 13X); 8. Sample3B, silica-zeolite membrane (Type 13X); 9. Sample 4A, S60 Silica-zeolitecomposite; 10. Sample 4B, S60 Silica-zeolite composite; 11. Sample 5Azeolite powder (Type 3A); 12. Sample 5B zeolite powder (Type 3A); 13.Blank; 14. Lambda DNA EcoRI/HindIII marker; and 15. 100 bp DNA ladder.The gel in FIG. 9A demonstrated that RNA has been purified usingdifferent types of zeolite (Type 3A or Type 13X) in membrane form eitherby itself or as a silica-zeolite composite membrane as well as asilica-zeolite composite in solution or zeolite powder. The gel in FIG.9B showed that no visible DNA co-purified with the RNA.

Example 12 Screening Zeolites

This example describes a method for screening zeolites for nucleic acidpurification. In particular, this example describes screening differentzeolites for the ability to leave RNA including small RNA in solutionand to remove DNA. The initial screen (part A) used a difficult lysatesample that has a lot of RNA, DNA and contaminants to challenge thezeolites. The second screen (part B) includes a subset of the zeolitesfrom part A, as well as an additional brand (Praxair) of MolecularSieves Type 13X which has the same synthesis source as Sigma catalog#3010. The second screen uses a typical lysate sample amount to examinethe effectiveness of the zeolites under less stringent conditions. Thedilution buffer employed was DB4 which is SV RNA Dilution Buffer Promega(Promega, 3M NaCl, 0.3M Na₃ citrate•2H₂O, 0.2% SDS, 0.1 mM EDTA (pH 8.0)and 0.0009% Blue dye (FD&C Blue #1)) with the final pH adjusted to pH4.0 with concentrated hydrochloric acid.

Part A TABLE 8 Tube # Zeolite Name Source Identification 1, 2 Molecularsieves, type 3A, Acros Organics, Fair Lawn, New Acros 3A <50 micronJersey cat#214795000, C.A.S. 308080-99-1 3, 4 Molecular sieves, type 3A,Aldrich, St. Louis, MO, cat Aldrich 3A powder, undried #233641-500 G,C.A.S. 308080- 99-1 5, 6 Molecular sieves, type 4A Acros cat#214805000,Acros 4A C.A.S. 70955-01-0 7, 8 Molecular sieves, type 4A Flukacat#69836 Fluka 4A  9, 10 Molecular sieves, Type 5A, Acros cat#214815000Acros 5A <50 micrometers C.A.S. 69912-79-4 11, 12 Molecular sieves, Type5A, Aldrich cat#233676-1 kg Aldrich 5A powder, undried 13, 14 Molecularsieves, Type 13x, Acros cat# 269255000, Acros 13X powder, <50 micronC.A.S. 63231-69-6 15, 16 Molecular sieves, Type 13x, AlfaAesar/Lancaster, Pelham, Alfa Aesar powder NH, cat#L06232 13X 17, 18Molecular sieves, UOP, Fluka, Switzerland, cat#69856 Fluka 13X Type 13x19, 20 Molecular sieves, Type 13x, Sigma Chemical Co., St. Louis, Sigma13X, <10 um, powder MO, cat#M3010-250 G 3010 21, 22 Molecular sieves,Type 13x, Sigma cat#283592-1 kg Sigma 13X #2 powder 23, 24 Molecularsieves, Aldrich cat#419095-100 G Aldrich Organophilic organophilic 25,26 Zeolite MP Biomedicals, LLC, Aurora, MP Bio zeolite OH, cat#193902

The following protocol was employed. 2 ml SV RNA Lysis Buffer (Promega)including 20 ul 48.7% beta mercaptoethanol (BME, Promega) per milliliterbuffer was added per 600 mg frozen bovine spleen and homogenized using arotor stator homogenizer. The homogenized lysate was divided into tubesand stored at minus 80° C. Tubes containing bovine spleen lysate wereremoved from the −80° C. freezer and thawed in water at 4° C. 2 mlaliquots of the bovine lysate were dispensed into 15 ml tubes. 4 ml DB4was added and 0.5 g zeolite (see the Table 8 above for the type ofzeolite added to the tube) was added to each 15 ml tube. The 15 ml tubeswere inverted and shaken well to mix the samples and then incubated in awater bath at 70° C. for 5 minutes. The 15 ml tubes were returned to arack at room temperature.

For each sample a PureYield Clearing Column (Promega) was inserted in 50ml polypropylene catch tube. One at a time, the 15 ml tubes wereinverted and shaken to mix and then immediately poured into thePureYield Clearing Column in the corresponding labeled tube. Theseclearing columns in catch tubes were centrifuged at 2000×g for 10minutes at 24° C. The Clearing Columns were discarded. 4 ml isopropylalcohol was added to the flow through in each tube and inverted andshaken 2-4 times to mix. This mixture was poured into a PureYieldBinding Column (Promega) in a labeled 50 ml tube and spun in acentrifuge at 2000×g at 24° C. Tubes 9 and 17-26 were centrifuged anadditional 3 minutes. Tubes 18 and 22-26 were centrifuged a third timefor an additional 3 minutes.

The binding columns were connected to a vacuum manifold (Promega). Thecolumns were washed with 20 ml and then 10 ml SV RNA Wash Solution(Promega) applying a vacuum. After all wash solution had flowed through,the vacuum was applied for an additional 3 minutes to dry the columnmembrane. The binding columns were transferred to clean 50 ml tubes. Toelute the RNA, 1 ml of nuclease-free water was applied to the bindingmembrane and incubated at 22° C. for 3 minutes with the tube capped. Thecolumns were spun in a centrifuge at 2000×g for 3 minutes.

To evaluate the eluates the samples were loaded on a gel. 10 ul of eachelution was incubated at 37° C. for two hours with 2.0 ul RNase ONE™ribonuclease enzyme (Promega) and 1.3 ul 10× RNase ONE™ buffer (Promega)to digest the RNA. The RNase ONE™-treated samples were stored at −20° C.2.5 ul of Blue/Orange 6× loading dye (Promega) was added to the thawedRNase ONE™-treated samples. 2 ul of Blue/Orange 6× loading dye was addedto 10 ul of untreated sample. Each lambda marker lane has 2 ul of lambdaDNA EcoRI/HindIII marker (Promega) mixed with 8 ul water and 2 ulBlue/Orange Loading Dye, 6×. Each 100 bp ladder lane has 2 ul 100 bp DNAladder (Promega) mixed with 8 ul water and 2 ul Blue/Orange Loading Dye,6×. 10 ul of lysate was mixed with 2 ul Blue/Orange Loading Dye, 6×. Thelysate and all samples with loading dye were loaded on a 1× TBE 1%agarose gel containing ethidium bromide.

The gel in FIG. 10A shows the presence of RNA in all samples, with somedifferences in the presence of small rRNA purified. The gel also showsdifferences in the amount of DNA carried through the purificationprocess. The most efficient zeolite tested here for capturing small rRNAis Sigma molecular sieves Type 13X (Sigma, cat#283592). The mostefficient zeolite tested for lack of DNA is the Acros molecular sievestype 3A. The molecular sieves Type 5A by Acros did slightly better thanType 5A by Aldrich but both show a certain amount of DNA as well as awhite opaque contaminant visible in the eluate in tubes 10, 11 and 12(small amount in tube 9). The top row and bottom rows in FIG. 10A arethe same samples loaded in the same order, except where noted. Thebottom row samples have been treated with RNase ONE™, while the bottomrow contains an additional lane with lysate that has not been treatedwith RNase. The lanes in FIG. 10A were loaded in the following order: 1.Lambda DNA EcoRI/HindIII marker, 2. Blank, 3. Acros 3A, 4. Acros 3A, 5.Aldrich 3A, 6. Aldrich 3A, 7. Acros 4A, 8. Acros 4A, 9. Fluka 4A, 10.Fluka 4A, 11. Acros 5A, 12. Acros 5A, 13. Aldrich 5A, 14. Aldrich 5A,15. Blank, 16. Lambda DNA EcoRI/HindIII marker, 17. 100 bp DNA ladder,18. Blank, 19. Acros Type 13X, 20. Acros Type 13X, 21. Alfa Aesar Type13X, 22. Alfa Aesar Type 13X, 23. Fluka Type 13X, 24. Fluka Type 13X,25. Sigma Type 13X, #3010, 26. Sigma Type 13X, #3010, 27. Sigma Type13X, #2, 28. Sigma Type 13X, #2, 29. Aldrich Organophilic, 30. AldrichOrganophilic, 31. MP Bio zeolite, 32. MP Bio zeolite, 33. Blank, 34.Lambda DNA EcoRI/HindIII marker, 35. Top: 100 bp DNA ladder, 35. Bottom:Blank, 36. Blank, 37. Top: Blank, 37. Bottom: Lysate.

Part B

For part B, the same protocol was followed as in part A with thefollowing exceptions: (1) The frozen bovine spleen lysate was dilutedwith an equal volume of SV RNA Lysis Buffer with BME; (2) The lysate anddilution buffer were mixed prior to addition of the powder zeolite; (3)The 70° C. incubation was for 3 minutes; (4) Only one centrifugation wasrequired for the clearing column step; (5) The RNase treatment ofsamples was a one-hour incubation only. TABLE 9 Tube # Zeolite NameSource Identification 1, 2 Molecular sieves, type 3A, Acros Organics,cat#214795000, Acros 3A <50 micrometers C.A.S. 308080-99-1 3, 4Molecular sieves, type 3A, Aldrich, cat #233641-500 G Aldrich 3A powder,undried 5, 6 Molecular sieves, Type 13x, Alfa Aesar/Lancaster,cat#L06232 Alfa Aesar 13X powder 7, 8 Molecular sieves, Type 13X,Praxair, Danbury, CT. part#MS-1329 Praxair 13X powder  9, 10 Molecularsieves, Type 13x, Sigma Chemical Co., cat#M3010- Sigma 13X, <10 um,powder 250 G 3010 11, 12 Molecular sieves, Type 13x, Sigma cat#283592-1kg Sigma 13X #2 powder

The gel in FIG. 10B shows that even the under less stringent conditionsof the second screen, the Aldrich type 3A still outperforms the AcrosType 3A for removing DNA while leaving RNA in solution. The Alfa AesarType 13X does better in the second screen for removing DNA contaminationthan in the more stringent screen of part A. As expected, the Praxairzeolite and the Sigma Type 13X cat #3010 (same description, synthesizedby the same company) behaved similarly by retaining the small rRNA andreducing the DNA contamination. The Sigma Type 13X cat#283592 gaveresults similar to the first screen and to the Aldrich Type 3A of thesecond screen. The lanes in FIGS. 10B (untreated samples) and 10C(RNase-treated samples) were loaded in the following order: 1. LambdaDNA EcoRI/HindIII marker; 2. Blank; 3. Acros 3A; 4. Acros 3A; 5. Aldrich3A; 6. Aldrich 3A; 7. Alfa Aesar 13X; 8. Alfa Aesar 13X; 9. Praxair 13X;10. Praxair 13X; 11. Sigma Type 13X, #3010; 12. Sigma Type 13X, #3010;13. Sigma Type 13X, #2; 14. Sigma Type 13X, #2; 15. Blank; 16. LambdaDNA EcoRI/HindIII marker; 17. 100 bp DNA ladder

Example 13 Generating Magnetic and Paramagnetic Zeolite Particles

This Example describes the production of Fe₂O₃-zeolite magneticparticles. 3 grams of zeolite (Aldrich cat# 233641) was placed into a 50ml tube, and 1 gram of Fe₂O₃ (Aldrich 31,006-9) was added. The contentswere mixed by vortexing, and 5 ml of 56% KOH was added, followed byadditional vortexing. The contents were poured into a Promega clearingcolumn inserted into a 50 ml plastic collection tube, and centrifuged at2000×g for 2 minutes in a swinging bucket rotor (all subsequent spins aswell). Then 3 ml of 1N HCl was added, and the column was centrifuged for2 minutes at 2000×g. 3 ml of 1N HCl was added to the column, and it wascentrifuged at 2000×g for 2 minutes. 5 ml of nuclease free water wasadded, and the column spun again for 2 minutes at 2000×g. Afterdiscarding the flowthrough from the collection tube, an additional 5 mlof nuclease free water was added to the column, and the column/tube wasspun again for 2 minutes at 2000×g.

The following tube was used as a balance in the centrifuge with theabove tube, and was used for MagneSil®-zeolite magnetic particleproduction. 10 ml of Promega MagneSil® (100 mg per ml) was added to aPromega clearing column, and the contents centrifuged 2000×g for 2minutes in a swinging bucket rotor (all subsequent spins as well). Theflowthrough was discarded, and the resulting 1 gm of MagneSil® was addedto a 50 ml plastic tube containing 3 gm of zeolite 3A (Aldrich cat#233641), and the contents were mixed by vortexing. Then 5 ml of 56% KOHwas added, followed by additional vortexing. The contents were pouredinto a Promega clearing column, inserted into a 50 ml collection tube,and centrifuged at 2000×g for 2 minutes. Then 3 ml of 1N HCl was added,and the column was centrifuged for 2 minutes at 2000×g. 3 ml of 1N HClwas added to the column, and it was centrifuged at 2000×g for 2 minutes.5 ml of nuclease free water was added, and the column spun again for 2minutes at 2000×g. After discarding the flowthrough from the collectiontube, an additional 5 ml of nuclease free water was added, and thecolumn spun again for 2 minutes at 2000×g.

The above Fe₂O₃-zeolite magnetic particles and the aboveMagneSil®-zeolite particles were each removed from their respectivecolumns and resuspended in 15 ml of nuclease free water, each, in clean50 ml plastic tubes. The tubes were magnetized for 30 seconds withmixing, so that most of the particles were captured by the magnet. Theremaining solution, for each particle type, was transferred to a clean50 ml tube which was then magnetized for 90 seconds with mixing. Theparticles magnetically captured were labeled “first cut” and theremaining particles were transferred to a clean 50 ml tube and thecontents magnetized for 5 minutes with occasional mixing. The particlesmagnetically captured in the final tube were labeled “second cut,” andthe remaining liquid was discarded. The particles produced by the“second cut” were lighter in color than the “first cut” particlesindicating a greater zeolite content on average. The initially capturedparticles were darker in color than the “first cut” particles. Usingthis method, magnetic (or paramagnetic) particles with greater zeolitecontent were enriched for by collecting particles with a slower magneticresponse time. Materials that were not magnetically responsive werediscarded at the end of the procedure.

Example 14 Fe₂O₃-Zeolite Magnetic Particle and MagneSil®-ZeoliteMagnetic Particle Purification

This Examples describes the use of Fe₂O₃ particles coated with zeoliteand MagneSil® particles coated with zeolite to purify RNA from lysate. AHEK 293 Dallas human cell culture was lysed with 4 ml SV RNA LysisBuffer (Promega) including 80 ul of 48.7% beta mercaptoethanol (BME,Promega). The flask was gently shaken with the lysis buffer to lyse thecells. The lysate was stored at −70° C.

The making of the zeolite-coated particles was previously described inExample 13. This experiment used the Fe₂O₃-zeolite magnetic particlesand the MagneSil®-zeolite magnetic particles initially captured withmagnetization, as well as the Fe₂O₃-zeolite magnetic particles “secondcut”. The magnetic-zeolite particles were suspended in DB4.36 at 100mg/ml. Zeolite Molecular Sieve Type 13X (Praxair) was also suspended inDB4.36 at 100 mg/ml. DB4.36 is 3M NaCl, 0.36M Na₃ citrate•2H₂O, 0.1 mMEDTA (pH 8.0), and 0.0009% Blue dye (FD&C Blue #1), adjusted to a finalpH of 4.0 with concentrated hydrochloric acid.

Twelve 50 ul aliquots of the human cell lysate were dispensed into a96-well microplate U-form (Greiner bio-one cat#650101) containing 100 ulDB4.36 per well, and mixed. 50 ul of zeolite or magnetic-zeoliteparticles (100 mg/ml) were added and mixed. The plate was heated in ahybridization oven at 70° C. for 15 minutes. The plate was then placedon a MagnaBot® 96 Magnetic Separation Device (Promega) to separate theparamagnetic particle from the cleared lysate. The cleared lysates weretransferred to clean wells in the 96-well plate. The Fe₂O₃-zeolitemagnetic particles (second cut) were transferred two subsequent times toclean wells on the MagnaBot® to separate the particles from the clearedlysate. The Type 13X zeolite samples (non-paramagnetic) were transferredto a Spin-X® centrifuge tube filter (Corning Inc. cat#8160) and spuntwice in a microcentrifuge at 8000 rpm for 1 minute each, plus anadditional spin at 12,000 rpm for 1 minute. The cleared lysate wastransferred to the 96-well plate. 10 ul of cleared lysate was removedfrom each sample for analysis.

100 ul of isopropanol was added to each well containing cleared lysatefrom Fe₂O₃-zeolite particles (main) and MagneSil®-zeolite particles(main). 60 ul of isopropanol was added to each well containing thecleared lysate from the Fe₂O₃-zeolite particles (second cut) and zeoliteType 13X, because the volume was just over half of the volume of thecleared lysate from Fe₂O₃-zeolite particles (main) and MagneSil®-zeoliteparticles (main). 10 ul of MagneSil Blue (Promega cat#A2201) was addedto each cleared lysate and mixed. The samples were incubated at 22° C.for 20 minutes. The plate was placed on the MagnaBot® magnetic separatorand after the paramagnetic particles separated, the lysate was removed.The paramagnetic particles were washed twice with 300 ul SV RNA WashSolution (Promega cat#Z309C) using the MagnaBot® magnetic separator. Thenucleic acid was eluted with 50 ul of nuclease-free water incubating for15 minutes at 50° C. on the heat block.

FIGS. 11A, 11B and 11C were generated as follows. 10 ul of each eluatewas incubated at 37° C. for 3 hours with 2.0 ul RNase ONE™ ribonucleaseenzyme (Promega part # M426C) and 1.3 ul 10× RNase ONE™ buffer (Promegapart# M217A) to digest the RNA. After the incubation the RNase treatedsamples were each mixed with 2.5 ul Blue/Orange Loading Dye, 6×(Promega) and 10 ul of untreated sample mixed with 2.0 ul Blue/OrangeLoading Dye, 6×. 10 ul of cleared lysate or 10 ul of lysate was mixedwith 2.0 ul Blue/Orange Loading Dye, 6×. Each lambda marker lane had 2ul of lambda DNA EcoRI/HindIII marker (Promega) mixed with 8 ul waterand 2 ul Blue/Orange Loading Dye, 6×. Each 100 bp ladder lane had 2 ul100 bp DNA ladder (Promega) mixed with 8 μl water and 2 ul Blue/OrangeLoading Dye, 6×. All samples with loading dye were loaded on a 1× TBE,1% agarose gel containing ethidium bromide.

FIGS. 11A and B demonstrated that RNA is purified using zeolite-coatedMagneSil® particle or zeolite-coated Fe₂O₃ particles. FIG. 11Bdemonstrated that the bands seen in FIG. 11A were removed with RNasetreatment and that DNA was not detected in the samples on the gel. FIG.11C demonstrated that the DNA was not present in the cleared lysate, butremoved from the lysate during the magnetic zeolite clearing step.

The samples were loaded in the same order for FIGS. 11A and 11B. Theonly differences were that 11B samples were treated with RNase ONE™ribonuclease while the 11A samples were untreated, and that 11B haslysate loaded in well 19 and an addition Lambda DNA Marker in well 20.The gels in FIGS. 11A and B were loaded in the following order: 1.Lambda DNA EcoRI/HindIII marker; 2. Blank; 3. MagneSil®-zeoliteparticles (main)-1; 4. MagneSil®-zeolite particles (main)-2; 5.MagneSil®-zeolite particles (main)-3; 6. Fe₂O₃-zeolite particles(main)-1; 7. Fe₂O₃-zeolite particles (main)-2; 8. Fe₂O₃-zeoliteparticles (main)-3; 9. Fe₂O₃-zeolite particles (second cut)-1; 10.Fe₂O₃-zeolite particles (second cut)-2; 11. Fe₂O₃-zeolite particles(second cut)-3; 12. Zeolite (Type 13X)-1; 13. Zeolite (Type 13X)-2; 14.Zeolite (Type 13X)-3; 15. Blank; 16. Lambda DNA EcoRI/HindIII marker;17. 100 bp DNA ladder; 18. Blank 19. Lysate (only in FIG. 11B); and 20.Lambda DNA EcoRI/HindIII marker (only in FIG. 11B). For FIG. 11C, whichshows the cleared lysate, and lysate, the sample loading order was asfollows, with two blank lanes between each sample and seven blank lanesbetween the last sample and the lysate: 1. MagneSil®-zeolite particles(main) cleared lysate-1; 2. MagneSil®-zeolite particles (main) clearedlysate-2; 3. MagneSil®-zeolite particles (main) cleared lysate-3; 4.Fe₂O₃-zeolite particles (main) cleared lysate-1; 5. Fe₂O₃-zeoliteparticles (main) cleared lysate-2; 6. Fe₂O₃-zeolite particles (main)cleared lysate-3; 7. Fe₂O₃-zeolite particles (second cut) clearedlysate-1; 8. Fe₂O₃-zeolite particles (second cut) cleared lysate-2; 9.Fe₂O₃-zeolite particles (second cut) cleared lysate-3; 10. Zeolite (Type13X) cleared lysate-1; 11. Zeolite (Type 13X) cleared lysate-2; 12.Zeolite (Type 13X) cleared lysate-3 13. Lysate.

Example 15 Purifying RNA from a RNA/DNA Mixture Using Magnetic ZeoliteParticles

This Example describes the use of magnetic zeolite particles to generatea purified RNA sample. A mixture of RNA and DNA was prepared bycombining 10 ul of 500 ug/ml kanamycin mRNA (Promega cat# C1381) with100 ul of 1 kb DNA ladder (Promega cat# G571A). Magnetic zeoliteparticles made (as described in Example 13) were added to 1.5 mlEppendorf tubes, 20 mg of particles per tube. To each tube was added (inorder): 10 ul of dilution buffer (DB4), 5 ul Lysis buffer, and 6 ul ofthe above RNA/DNA mixture. Tubes were capped, mixed and heated for 5minutes at 70° C., then cooled to 21° C. for 5 minutes, and then placedon a magnetic separation stand for 10 minutes. 5 ul of each magneticallyseparated solution was added per well on a 1% agarose gel, as shown inFIG. 12. Due to considerable salt/buffer in many samples, there is a“salt front” that curves sample migration in the gel. The lanes in FIG.12 were loaded as follows: 1. Fe₂O₃ control (no zeolite coating); 2.Fe₂O₃-zeolite “main;” 3. Fe₂O₃-zeolite “first cut;” 4. Fe₂O₃-zeolite“second cut,” 5. RNA/DNA mix, no particle treatment; 6. kanamycin mRNAstandard; 7 MagneSil-zeolite “main,” 8. MagneSil-control, not coatedwith zeolite; 9. blank; 10. MagneSil-zeolite “first cut;” 11MagneSil-zeolite “second cut;” and 12. blank. The gel in FIG. 12 showsthat magnetic zeolites removed DNA from a RNA/DNA mixture, usingmagnetic separation to purify RNA away from DNA.

Example 16 The Effect of GTC on Removal of DNA from a RNA/DNA Mixture

This Example describes the effect of guanidine on RNA purification withzeolites.

A mixture of RNA and DNA was prepared by combining 10 ul of 500 ug/mlkanamycin mRNA (Promega cat# C1381) with 25 ul of 1 kb DNA ladder(Promega cat# G571A). 100 mg of zeolite 13X (Sigma-Aldrich cat# 283592)was added per column, for two columns (Costar cat# 8169, Corning, N.Y.(containing a 0.22 micron nylon membrane)), and 100 mg of zeolite 3A(Aldrich cat# 233641) per Costar 8169 column, for two columns. Allcolumns were placed in 1.5 ml microfuge tubes.

To columns 13X-DB4-only and 3A-DB4-only, 100 ul of dilution buffer DB4(see Example 3) was added. For samples 13α-plus-Lysis and 3A-plus-Lysis,100 ul DB4 and 50 ul of Lysis solution (4M guanidine thiocyanate, 10 mMTris, pH 7.5, with beta-mercaptoethanol added separately to a finalconcentration of 0.974% (v/v)), was added per column. The contents ofall 4 columns were mixed, and 5 ul of the RNA/DNA mix (above) was addedper column. The tubes were capped, and the tubes were incubated at 70°C. for 3 minutes, then cooled to 21° C. for 5 minutes. The tubes werecentrifuged at 8000×g for 60 seconds. 10 ul of the flowthroughs wereloaded per well in a 1% agarose non-denaturing gel. The results areshown in FIG. 13. The lanes in FIG. 13 were loaded in the followingorder: 1. and 2. were blank; 3. 1/10^(th) diluted RNA/DNA mix standard;4. blank; 5. zeolite 13X plus Lysis; 6. blank; 7. 13X DB4 only; 8.blank; 9. undiluted RNA/DNA mix standard; 10.50% diluted RNA/DNA mixstandard; 11. blank; 12. zeolite 3A-plus-Lysis; 13. blank; and 14.3A-DB4-only. The gel in FIG. 13 shows that zeolite 13X removed DNA indilution buffer BD4 only, as well as in the DB4-plus-Lysis sample.Zeolite 3A partially removed DNA, leaving RNA, and the DB4-plus Lysissample showed no visually detectable DNA, leaving the RNA in theflowthrough. As such, this example shows the purification of RNA(removal of DNA) with zeolites and dilution buffer alone, and enhancedpurification of RNA with zeolites and a buffer containing guanidine.

Example 17 Dilution Buffer pH Range, Used with Liver Lysates

This example describes various pH ranges for citrate buffer that areeffective for purifying RNA with zeolites. 5.71 g frozen bovine livertissue was homogenized in 114.2 ml SV RNA Lysis Buffer (Promega cat#Z3051), including 2.28 ml of 48.7% beta mercaptoethanol (BME, Promegapart#Z523A) using a rotor stator homogenizer. All the tubes used in thisexample were polypropylene screw-cap centrifuge tubes. 2 ml aliquots ofthe bovine liver homogenate (lysate) were dispensed into 15 ml tubes andthe remainder of the lysate was stored at −80° C. Four (4.0) ml bufferwas added to each of the tubes with lysate. The citrate buffer was 3MNaCl, 0.36M Na₃ citrate•2H₂O adjusted to a final pH ranging from 2.9 to6.4 with concentrated hydrochloric acid. Two negative control tubes hadno buffer added to the lysate. All 15 ml tubes were inverted 3-4 timesand shaken to mix the samples. The tubes were incubated in a water bathat 70° C. for 5 minutes and then returned to a rack at room temperatureto cool for 5 minutes.

For each sample a PureYield Clearing Column (Promega) was inserted in 50ml polypropylene catch tube (caps discarded). One at a time, the 15 mltubes were inverted and shaken to mix and then immediately poured intothe PureYield Clearing Column in the corresponding labeled tube. Theseclearing columns in catch tubes were centrifuged at 2000×g for 10minutes at 24° C. The Clearing Columns were discarded. 4 ml isopropylalcohol was added to the flow through in each tube and inverted andshaken well to mix. This mixture was poured into a correspondinglylabeled PureYield™ Binding Column (Promega cat #Z3091) attached to avacuum manifold and vacuum was applied. The negative control tubeswithout buffer clogged as well as tube 2.9B (citrate buffer at pH 2.9)and so were not processed beyond this step.

After the mixture passed through the column, 20 ml and then 10 ml SV RNAWash solution was added to the binding column with the vacuum appliedeach time until there was no standing wash solution on the columnmembrane. The vacuum was applied for an additional 3 minutes to dry themembrane. The binding columns were transferred to clean 50 ml tubes. Toelute the RNA, 1 ml of nuclease-free water was applied to the bindingmembrane and incubated at 22° C. for 2 minutes. The columns werecentrifuged at 2000×g for 3 minutes at 24° C.

FIGS. 14A and 14B were generated as follows. 10 ul of each sample wasincubated at 37° C. for 1.5 hours with 2.0 ul RNase ONE™ ribonucleaseenzyme (Promega) and 1.3 ul 10× RNase ONE™ buffer (Promega) to digestthe RNA. After the incubation the RNase treated samples were each mixedwith 2.5 ul Blue/Orange Loading Dye, 6× (Promega) and 10 ul of untreatedsample mixed with 2.0 ul Blue/Orange Loading Dye, 6×. Each lambda markerlane had 2 ul of lambda DNA EcoRI/HindIII marker (Promega) mixed with 8ul water and 2 ul Blue/Orange Loading Dye, 6×. Each 100 bp ladder lanehad 2 ul 100 bp DNA ladder (Promega) mixed with 8 ul water and 2 ulBlue/Orange Loading Dye, 6×. All samples with loading dye were loaded ona 1× TBE, 1% agarose gel containing ethidium bromide (Cambrex).

The gel in FIG. 14A (untreated samples) was loaded in the followingorder: 1. Lambda DNA EcoRI/HindIII marker; 2. Blank; 3. pH 2.9A; 4.blank; 5. pH 3.4A; 6. pH 3.4B; 7. pH 4.1A; 8. pH 4.1B; 9. pH 4.6A; 10.pH 4.6B; 11. pH 5.0A; 12. pH 5.0B; 13. pH 5.5A; 14. pH 5.5B; 15. pH6.0A; 16. pH 6.0B; 17. pH 6.4A; 18. pH 6.4B; 19. Lambda DNAEcoRI/HindIII marker; and 20. 100 bp DNA ladder. The gel in FIG. 14B(RNase-treated samples) was loaded in the following order: 1. Lambda DNAEcoRI/HindIII marker; 2. 100 bp DNA ladder; 3. Blank; 4. pH 2.9A,Treated with RNase; 5. pH 3.4A, Treated with RNase; 6. pH 3.4B, Treatedwith RNase; 7. pH 4.1A, Treated with RNase; 8. pH 4.1B, Treated withRNase; 9. pH 4.6A, Treated with RNase; 10. pH 4.6B, Treated with RNase;11. pH 5.0A, Treated with RNase; 12. pH 5.0B, Treated with RNase; 13. pH5.5A, Treated with RNase; 14. pH 5.5B, Treated with RNase; 15. pH 6.0A,Treated with RNase 16. pH 6.0B, Treated with RNase; 17. pH 6.4A, Treatedwith RNase; 18. pH 6.4B, Treated with RNase; 19. Blank; and 20. LambdaDNA EcoRI/HindIII marker. FIG. 14A demonstrates that RNA has beenpurified away from DNA at a citrate buffer pH ranging from 3.4 to 5.0,while citrate buffer pH range from 5.5 to 6.4 purifies both RNA and DNA.FIG. 14B demonstrates that no visible DNA co-purified with the RNA at pH3.4-5.0, while DNA is present at pH 5.5-6.4.

Example 18 RNA Purification Employing Various pH Ranges

This example describes the removal of DNA from a mixture of RNA and DNAusing zeolites and citrate buffer at various pH ranges. A mixture of RNAand DNA was prepared by adding 150 ul of 1 kb DNA ladder (Promegacat#G571A) to 10 ug of kanamycin mRNA (Promega cat# C1381). 10 mg ofzeolite 13X ((Sigma-Aldrich cat# 283592) was added per column to 8columns (Costar #8169) nested in 1.5 ml tubes, each column containing100 ul of 0.6M citrate buffer at a different pH: (1) 5.0, (2) 5.0, (3)5.1, (4) 5.2, (5) 5.2 plus an additional 50 ul of Lysis buffer (4Mguanidine thiocyanate, 10 mM Tris pH 7.5, 97% beta-mercaptoethanol), (6)pH 5.3, (7) pH 5.5 and (8) pH 6.4. 10 ul of the above RNA/DNA mix wasadded per column, and the tubes capped and incubated at 70° C. for 3minutes (except sample (1) which was kept at room temperature). Sampleswere incubated at 22° C. for 5 minutes, then centrifuged at 8000×g for 2minutes. 10 ul of the flowthrough was run per well on a 1% agarose, TBEnon-denaturing gel. The resulting gel photo is shown below in FIG. 15.The lanes in FIG. 15 were loaded as follows: 1. RNA/DNA mixture,standard; 2. 0.6M citrate pH 5.0, no 70° C. incubation; 3. blank; 4.0.6M citrate pH 5.0, with 70° C. incubation; 5. blank; 6. 0.6M citratepH 5.1; 7. blank; 8. RNA/DNA mixture, standard; 9.1 kb DNA ladder,standard; 10. 0.6M citrate pH 5.2; 11. blank; 12. 0.6M citrate pH 5.2plus lysis solution; 13. blank; 14. 0.6M citrate pH 5.3; 15. blank; 16.0.6M citrate pH 5.5; 17. 1 kb DNA ladder, standard; 18. 0.6M citrate pH6.4; 19. blank; 20. 10 ul sample of lane 6 plus 1 ul of DNA/RNA mix.This result has demonstrated removal of DNA without RNA removal using0.6M citrate buffers with pH ranging from pH 5.0 to pH 5.3.

Example 19 Removal of DNA from Protein Solutions

This example describes the removal of DNA from protein solutions usingzeolites. 10 mg of zeolite 13X ((Sigma-Aldrich cat# 283592) was addedper column to 2 columns (Costar #8169) nested in 1.5 ml tubes. 40 ul of0.6M sodium citrate buffer, pH 4.6 was added to each column, and mixed.Then 10 ul of 1 kb DNA ladder (Promega cat#G571A), and 5 ul of CelluACE™(Promega cat# FF3800) enzyme mixture was added and the contents mixed.Two columns (3 and 4) with identical contents to 1 and 2 above wereprepared, except that they did not contain zeolite. All 4 tubes werecapped and incubated at 50° C. for 20 minutes, then incubated at 21° C.for 5 minutes. The columns were microfuged at 8000×g for 2 minutes. 10ul of each sample flowthrough was run per well, in a 1% TBEnon-denaturing agarose gel. As shown in the FIG. 16 gel photo, thesamples without zeolite have retained the DNA, but those containingzeolite have no visible DNA remaining in the sample. CelluACE activitywas measured and the samples containing zeolite showed 866 units ofactivity, while those without zeolite (still containing DNA) showed 1306units of activity. The resulting gel photo is shown in FIG. 16. Thelanes in FIG. 16 were loaded as follows: 1. CelluACE plus zeolite A; 2.CelluACE plus zeolite B; 3. CelluACE, no zeolite A; and 4. CelluACE, nozeolite B.

Example 20 Isolation of Total RNA from Cow Heart Using an AcetateDilution Buffer

This example describes the isolation of total RNA from cow heart tissueusing an acetate dilution buffer. A cow heart lysate was prepared byhomogenizing frozen tissue in 4° C. Lysis Buffer (4M guanidinethiocyanate, 10 mM Tris, pH 7.5, with beta-mercaptoethanol addedseparately to a final concentration of 0.974% (v/v)) with a PRO 200rotor/stator homogenizer (PRO Scientific, Inc., Oxford, Conn.). Thelysate was prepared at a concentration of 300 mg/ml (wet weight) and wasstored in 10 ml aliquots at −70° C., until use. The frozen lysate wasthawed at 4° C. The lysate was diluted to 150 mg/ml by adding an equalvolume of 4° C. Lysis Buffer. Two 1 ml aliquots (150 mg of cow heart perisolation) were dispensed into plastic, 15 ml, screw capped tubes. Anadditional 1 ml of 4° C. lysis buffer was added to increase the finalvolume to 2 ml. Four ml of Acetate dilution buffer, designated “DB4.6A”(0.6M Na acetate (pH adjusted to 4.01 with glacial acetic acid), 3MNaCl; adjusted to a final pH of 4.0 with 10N NaOH), was added to eachsample, mixed by inversion 3-4 times and then vortexed untilhomogeneous. One ml of clearing agent (2M NaCl, 0.1 mM EDTA (pH 8.0),0.45 g/ml Molecular Sieves, type 13X (zeolite)) was added to each tube,mixed by inversion 2-3 times and then vortexed until homogeneous. Thetubes were inverted 2-3 times a second time to resuspend the clearingagent, incubated at 70° C. in a hybridization oven for 5 minutes andthen placed at ambient temperature (23-24° C.) for 5 minutes to cool.

The following steps were performed at ambient temperature (23-24° C.).Each mixture was shaken vigorously, vortexed and poured into a Promegaclearing column, nested in a 50 ml collection tube. The columns werecentrifuged in a swinging bucket rotor at 2,000×g, 23° C. for 10minutes. The cleared lysates, containing RNA, were captured in 50 mlcollection tubes. The sample debris, clearing agent and DNA werecaptured by the clearing column membrane and were discarded with theclearing columns. Four ml of isopropanol was added to each tube ofcleared lysate and mixed by swirling the tube. Each mixture was appliedto a Promega binding column, and attached to a vacuum manifold. A vacuumof approximately 15 in. Hg was applied to the columns. Each samplepassed through the column, leaving the RNA bound to the binding columnmembrane. The membranes were washed twice with 20 ml and then 10 ml ofwash solution (60 mM potassium acetate, 10 mM Tris, pH 7.5, 60% ethanol)to remove impurities and salts. The membranes were vacuum dried for atleast 3 minutes on the vacuum manifold. The binding columns weretransferred to 50 ml collection tubes for elution. One ml ofnuclease-free water was applied to each membrane and incubated for 2-3minutes at 23-24° C. The column assemblies were centrifuged, using aswinging bucket rotor, at 2,000×g for 3 minutes to collect the purifiedtotal RNA. The purified total RNA samples were analyzed byspectrophotometry and agarose gel analysis. Total RNA yields weredetermined by absorbance at 260 nm. The results are shown in table 10below and FIGS. 17A and 17B. TABLE 10 Total RNA Yield from Cow HeartUsing an Acetate Dilution Buffer. Total RNA Yield Sample ID (μg) H150 118.4 H150 2 15.6

FIG. 17A shows a gel Analysis of purified total RNA from cow heart usingan acetate dilution buffer. RNA samples were analyzed by electrophoresison a native, 1% agarose, 1× TBE gel, stained with ethidium bromide. Tenμl of each sample was loaded per lane. The image was collected using anAlpha Innotech FluorChem™ Imaging System. The lanes of the gel are asfollows: Lane 1: H150 1; Lane 2: H150 2. Lane M was Promega's 1 kb DNALadder (Cat.# G5711), 10,000 bp, 8,000 bp, 6,000 bp, 5,000 bp, 4,000 bp,3,000 bp, 2,500 bp, 2,000 bp, 1,500 bp, 1,000 bp, 750 bp, 500 bp, 253 bpand 250 bp. Representative size markers are indicated. The undenatured23S ribosomal RNA, 16S ribosomal RNA and small RNAs are indicated byarrows.

FIG. 17B shows the results of a DNA contamination assay of total RNApurified from cow heart using an acetate dilution buffer. Purified totalRNA samples were digested with Promega's RNase ONE™ Ribonuclease (Cat.#M4265). A 15 μl digestion mix, containing 3 μl of 10× Reaction Buffer, 9μl of Nuclease-Free Water and 3 μl of RNase ONE™ Ribonuclease (5-10u/μl) was mixed with 15 μl of each total RNA sample. The digestions wereincubated at 37° C. for 1 hour and then held at 4° C., until analyzed.The digests were analyzed by electrophoresis on a native, 1% agarose, 1×TBE gel, stained with ethidium bromide. Ten μl of each reaction wasloaded per lane. The image was collected using an Alpha InnotechFluorChem™ Imaging System. Lanes of the gel are as follows: Lane 1: H1501; Lane 2: H150 2. Lane M was Promega's 1 kb DNA Ladder (Cat.# G5711),10,000 bp, 8,000 bp, 6,000 bp, 5,000 bp, 4,000 bp, 3,000 bp, 2,500 bp,2,000 bp, 1,500 bp, 1,000 bp, 750 bp, 500 bp, 253 bp and 250 bp.Representative size markers are indicated. The undenatured 23S ribosomalRNA, 16S ribosomal RNA and small RNAs are indicated by arrows.

Example 21 The Effect of Heat on Removal of DNA From a RNA/DNA Mixture

This Example describes the effect of heat on RNA purification withzeolites. Frozen bovine spleen tissue was homogenized with 1.0 ml LysisSolution per 300 mg tissue using a rotor stator homogenizer. The LysisSolution was SV RNA Lysis Buffer (Promega Cat. #Z3051) with betamercaptoethanol (BME, Promega Part #Z523A) added to a finalconcentration of 1%. The homogenate was divided into tubes and stored atminus 80° C. All the tubes used in this example were polypropylenescrew-cap centrifuge tubes.

Tubes containing homogenized bovine spleen were removed from the −80° C.freezer and thawed in water at 4° C. The bovine spleen homogenates werecombined and then diluted with an equal volume of the Lysis Solution. 2ml aliquots of the diluted bovine spleen homogenate were dispensed intofourteen 15 ml tubes. Four (4.0) ml DB4.36-EDTA (minus EDTA) was addedto each of tubes 1-6. DB4.36—EDTA is 3M NaCl and 0.36M Na₃ citrate•2H₂Oadjusted with concentrated hydrochloric acid to a final pH of 4.0. Four(4.0) ml DB4.36 (pH=3.8) was added to each of tubes 7-9. DB4.36 (pH=3.8)was made by adjusting the pH of DB4.36—EDTA with concentratedhydrochloric acid to a pH of 3.82. Four (4.0) ml DB4.36+EDTA was addedto each of tubes 10-14. DB4.36+EDTA is 3M NaCl, 0.36M Na₃ citrate•2H₂O,0.1 mM EDTA (pH 8.0), and 0.0009% Blue dye (FD&C Blue #1) adjusted withconcentrated hydrochloric acid to a final pH of 4.0.

To tubes 1-3 was added 1 ml zeolite mixture, which consisted of 2M NaCl,0.1 mM EDTA (pH 8.0) with zeolite type 13X added at a concentration of0.42 g/ml. The 3 tubes were inverted 3-4 times and shaken to mix thesamples. The 3 tubes were incubated in a water bath at 70° C. for 3minutes and then returned to room temperature.

Tubes 4-9 were inverted 34 times and shaken to mix the samples. Tubes4-9 were incubated in a water bath at 70° C. for 3 minutes and thenreturned to room temperature. After cooling for 5 minutes, 1 ml zeolitemixture which consisted of 2M NaCl, 0.1 mM EDTA (pH 8.0) with zeoliteadded at a concentration of 0.42 g/ml, was added to each of tubes 4-9and then inverted 3-4 times and shaken to mix the samples.

To tubes 10-12 was added 1 ml zeolite mixture, which consisted of 2MNaCl, 0.1 mM EDTA (pH 8.0) with zeolite added at varying concentrations:tube 10 had 0.40 g/ml, tube 11 had 0.43 g/ml, and tube 12 had 0.50 g/ml.Tubes 10-12 were inverted 3-4 times and shaken to mix the samples. Tubes10-12 were incubated in a water bath at 70° C. for 3 minutes and thenreturned to room temperature.

Tubes 13 and 14 were inverted 3-4 times and shaken to mix the samples.Both tubes were incubated in a water bath at 70° C. for 3 minutes andthen returned to a rack at room temperature. After cooling for 5minutes, 0.50 g zeolite powder was added to each of tubes 13 and 14 andthen inverted 3-4 times and shaken to mix the samples.

For each sample a PUREYIELD Clearing Column (Promega) was inserted in a50 ml polypropylene catch tube (cap discarded) labeled C1-C14. One at atime, the 15 ml tubes 1-14 were inverted and shaken to mix and thenimmediately poured into the PUREYIELD Clearing Column in thecorresponding labeled tube. These clearing columns in catch tubes werecentrifuged at 3000×g for 10 minutes at 22° C. Four (4) ml isopropylalcohol was added to the flow through in each tube and inverted 2-4times to mix. This mixture was poured into a PUREYIELD Binding Column(Promega Cat. # A245), labeled B1-B14, in a 50 ml tube and spun in acentrifuge at 3000×g at 22° C. for 10 minutes.

Each binding column was transferred to a fresh 50 ml tube. 20 ml SV RNAWash Solution (Promega Cat. #Z3091) was added each binding column andcentrifuged at 3,000×g for 6 minutes. The flow through was discarded.Then 10 ml SV RNA Wash Solution was added to each binding column andcentrifuged at 3,000×g for 6 minutes. The flow through was discarded.Each binding column was transferred to clean 50 ml tubes.

To elute the RNA, 1 ml of Nuclease-Free Water (Promega Cat. # P119C) wasapplied to each binding membrane and incubated at 22° C. for 1 minute.The columns were centrifuged at 3000×g for 2 minutes (RNA elution 1).Each column was transferred to a fresh 50 ml tube and the elution wasrepeated (RNA elution 2).

RNase ONE™ Ribonuclease Treatment

For each of the RNA elution 1 samples listed above, 10 ul of sample wasadded to 1.2 ul of RNase ONE 10× Buffer (Promega) in a PCR strip tubeand then 1.0 ul of RNase ONE Ribonuclease enzyme (Promega) was added.The strip tube was capped and incubated at 37° C. for about one hour.TABLE 11 zeolite added before or tube # Dilution buffer zeolite (g)after heat incubation 1-3 DB4.36 − EDTA 0.42 before 4-6 DB4.36 − EDTA0.42 after 7-9 DB4.36 (pH 3.8) 0.42 after 10 DB4.36 + EDTA 0.40 before11 DB4.36 + EDTA 0.43 before 12 DB4.36 + EDTA 0.50 before 13-14 DB4.36 +EDTA 0.50 powder after

To evaluate the eluates the samples were loaded on a 1% agarose, 1× TBEgel containing ethidium bromide. 10 ul of each elution 1 or elution 2was added to 2 ul Blue/Orange Loading Dye, 6× (Promega). 2.5 ulBlue/Orange Loading Dye, 6× was added to the RNaseONE-treated samples.Each lambda marker lane had 2 ul of lambda DNA EcoRI/HindIII marker(Promega Cat. # G1731) mixed with 9 ul water and 2 ul Blue/OrangeLoading Dye, 6×. Each 100 bp DNA Ladder had 3 ul of 100 bp DNA Ladder(Promega Cat. # G2101) mixed with 7 ul water and 2 ul Blue/OrangeLoading Dye, 6×.

The lanes in the top row of FIG. 18 were loaded in the following order(see Table 11 for description): 1. Lambda DNA EcoRI/HindIII marker; 2.100 bp DNA Ladder; 3. Blank; 4. Sample 1, elution 1; 5. Sample 2,elution 1; 6. Sample 3, elution 1; 7. Sample 4, elution 1; 8. Sample 5,elution 1; 9. Sample 6, elution 1; 10. Sample 7, elution 1; 11. Sample8, elution 1; 12. Sample 9, elution 1; 13. Sample 10, elution 1; 14.Sample 11, elution 1; 15. Sample 12, elution 1; 16. Sample 13, elution1; 17. Sample 14, elution 1; 18. Blank 19. Lambda DNA EcoRI/HindIIImarker; 20. 100 bp DNA Ladder; 21. Blank; 22. Sample 1, RNase treated;23. Sample 2, RNase treated; 24. Sample 3, RNase treated; 25. Sample 4,RNase treated; 26. Sample 5, RNase treated; 27. Sample 6, RNase treated;28 Sample 7, RNase treated; 29. Sample 8, RNase treated; 30. Sample 9,RNase treated; 31. Sample 10, RNase treated; 32. Sample 11, RNasetreated; 33. Sample 12, RNase treated; 34. Sample 13, RNase treated; 35.Sample 14, RNase treated; and 36. Lambda DNA EcoRI/HindIII marker.

The lanes in the bottom row of FIG. 18 were loaded in the followingorder (see Table 11 for description): 1. Lambda DNA EcoRI/HindIIImarker; 2. 100 bp DNA Ladder; 3. Blank; 4. Blank; 5. Sample 1, elution2; 6. Sample 2, elution 2; 7. Sample 3, elution 2; 8. Sample 4, elution2; 9. Sample 5, elution 2; 10. Sample 6, elution 2; 11. Sample 7,elution 2; 12. Sample 8, elution 2; 13. Sample 9, elution 2; 14. Sample10, elution 2; 15. Sample 11, elution 2; 16. Sample 12, elution 2; 17.Sample 13, elution 2; 18. Sample 14, elution 2; 19. Blank 20. Lambda DNAEcoRI/HindIII marker; and 21. 100 bp DNA Ladder.

The gel in FIG. 18 shows that when zeolite was added before the heatingstep there was no visible DNA in the eluate. In contrast, when thezeolite was added after the heat step when samples had cooled for 5minutes, DNA was visible in the eluate.

Example 22 Heating the Reaction at Different Temperatures

This example describes the use of heat at different temperatures toimprove the binding of DNA to zeolites. Frozen bovine spleen tissue washomogenized with 1.0 ml Lysis Solution per 150 mg tissue using a rotorstator homogenizer. The Lysis Solution was SV RNA Lysis Buffer (PromegaCat. #Z3051) with beta-mercaptoethanol added to a final concentration of1%. The homogenate was divided into tubes and stored at minus 80° C. Allthe tubes used in this example were polypropylene screw-cap centrifugetubes.

Tubes containing homogenized bovine spleen were removed from the −80° C.freezer and thawed in water at 4° C. 2 ml aliquots of the bovine spleenhomogenate were dispensed into fourteen 15 ml tubes. Four (4.0) mlDB4.36 was added to each of the tubes 1-14. DB4.36 is 3M NaCl, 0.36M Na₃citrate•2H₂O, 0.1 mM EDTA (pH 8.0), and 0.0009% Blue dye (FD&C Blue #1)adjusted with concentrated hydrochloric acid to a final pH of 4.0.

To tubes 1-12 was added 1 ml zeolite mixture, which consisted of 2MNaCl, 0.1 mM EDTA (pH 8.0) with zeolite Type 13X added at aconcentration of 0.45 g/ml. Tubes 1-12 were inverted 3-4 times andshaken to mix the samples. Tubes 13 and 14 had no zeolite mixture added.Tubes 1-14 were incubated in a water bath for 3 minutes and thenreturned to a rack at room temperature. The temperature of the waterbaths were as follows: tubes 1-3 at 10° C., tubes 4-6 at 21° C., tubes7-9 at 37° C., and tubes 10-12 at 70° C.

For each sample a PUREYIELD Clearing Column (Promega) was inserted in a50 ml polypropylene catch tube (cap discarded) labeled C₁-C₁₄. One at atime, the 15 ml tubes 1-14 were inverted and shaken to mix and thenimmediately poured into the PUREYIELD Clearing Column in thecorresponding labeled tube. These clearing columns in catch tubes werecentrifuged at 2000×g for 10 minutes at 22° C. Tubes 13 and 14, withoutzeolite, did not have all the liquid pass through the clearing column.Four (4) ml isopropyl alcohol was added to the flow through in each tubeand inverted 2-4 times to mix. This mixture was poured into a PUREYIELDBinding Column (Promega cat# A245), labeled B1-B14, in a 50 ml tube andspun in a centrifuge at 2000×g at 22° C. for 10 minutes. Tubes 13 and 14did not have all the liquid pass through the binding column.

Each binding column was transferred to a fresh 50 ml tube. 20 ml SV RNAWash Solution (Promega Cat #Z3091) was added to the each binding columnand centrifuged at 2,000×g for 5 minutes. The flow through wasdiscarded. Then 10 ml SV RNA Wash Solution was added to the bindingcolumn and centrifuged at 2,000×g for 10 minutes. The flow through wasdiscarded. Each binding column was transferred to clean 50 ml tubes.

To elute the RNA, 1 ml of nuclease-free water was applied to the bindingmembrane and incubated at 22° C. for 2 minutes. The columns werecentrifuged at 2000×g for 3 minutes (RNA elution 1). Each column wastransferred to a fresh 50 ml tube and the elution with 1 mlnuclease-free water was repeated (RNA elution 2).

For ribonuclease digestion, 10 ul of each of the RNA elution 1 sampleslisted above was added to 1.2 ul of RNase ONE 10× Buffer (Promega Part#217A) in a PCR strip tube and then 2.0 ul of RNase ONE Ribonucleaseenzyme (Promega part # M4261) was added. The strip tube was capped andincubated at 37° C. for about an hour.

To evaluate the eluates the samples were loaded on a 1% agarose, 1× TBEgel containing ethidium bromide. 10 ul of each elution 1 or elution 2was added to 2 ul Blue/Orange Loading Dye, 6× (Promega). 2.5 ulBlue/Orange Loading Dye, 6× was added to the RNaseONE-treated samples.Each lambda marker lane had 2 ul of lambda DNA EcoRI/HindIII marker(Promega cat# G1731) mixed with 9 ul water and 2 ul Blue/Orange LoadingDye, 6×. Each 100 bp DNA Ladder had 3 ul of 100 bp DNA Ladder (Promegacat# G2101) mixed with 7 ul water and 2 ul Blue/Orange Loading Dye, 6×.

In FIG. 19 the top row and bottom rows in the gel were loaded in thesame order as listed below, except where noted. The bottom row sampleswere treated with RNaseONE™. The lanes were as follows: 1. Lambda DNAEcoRI/HindIII marker; 2. 100 bp DNA Ladder; 3. Blank; 4. Sample 1, 10°C., elution 1; 5. Sample 2, 10° C., elution 1; 6. Sample 3, 10° C.,elution 1; 7. Sample 4, 21° C., elution 1; 8. Sample 5, 21° C., elution1; 9. Sample 6, 21° C., elution 1; 10. Sample 7, 37° C., elution 1; 11.Sample 8, 37° C., elution 1; 12. Sample 9, 37° C., elution 1; 13. Sample10, 70° C., elution 1; 14. Sample 11, 70° C., elution 1; 15. Sample 12,70° C., elution 1; 16. Sample 13, no zeolite, elution 1; 17. Sample 14,no zeolite, elution 1; 18. Blank; 19. Lambda DNA EcoRI/HindIII marker;20. Top: 100 bp DNA Ladder; 20. Bottom: Blank; 21. Blank; 22. Sample 1,10° C., elution 2; 23. Sample 2, 10° C., elution 2; 24. Sample 3, 10°C., elution 2; 25. Sample 4, 21° C., elution 2; 26. Sample 5, 21° C.,elution 2; 27. Sample 6, 21° C., elution 2; 28. Sample 7, 37° C.,elution 2; 29. Sample 8, 37° C., elution 2; 30. Sample 9, 37° C.,elution 2; 31. Sample 10, 70° C., elution 2; 32. Sample 11, 70° C.,elution 2; 33. Sample 12, 70° C., elution 2; 34. Sample 13, no zeolite,elution 2; 35. Sample 14, no zeolite, elution 2; 36. Blank; 37. LambdaDNA EcoRI/HindIII marker; 38. Top: 100 bp DNA Ladder; and 38. Bottom:Blank.

The gel in FIG. 19 showed the most DNA present in samples 13 and 14(lanes 16 and 17) which did not have zeolite. There was also an intenseDNA band in elution 1 and elution 2 of samples 1-3 (lanes 4-6 and 22-24)with the “heat” incubation at 10° C. When the heat incubation was 21°C., DNA was visible on the gel in elution 1 of sample 6 (lane 9) but notelution 2 (lane 27), and not in either elution of samples 4 or 5 (lanes7, 8, 25, and 26). The DNA band was much less intense in sample 6elution 1 than the 10° C. incubation eluates or the “no zeolite”eluates. No DNA was visible in the elutions of samples heated at 37° C.or 70° C. (lanes 10-15 and 28-33). These results showed that at roomtemperature zeolite bound DNA and that increasing the temperaturegenerally improved the amount of DNA bound, and therefore removal of DNAfrom the final purified RNA improved.

Example 23 Zeolite 13X Binding of Oligonucleotides, and Elution therefrom

This example describes the binding of zeolite 13X to a mixture of doublestranded 35 bp DNA, single stranded 35 base DNA, single stranded 30 baseDNA, 25 bp double stranded RNA, 25 base single stranded RNA and 21 basesingle stranded RNA, and elutions of the bound oligonucleotides. Thenucleic acid bound to the zeolite particles was separated from theremaining aqueous phase by placing the zeolite particles in a Corning0.22% nylon membrane Spin-X column, followed by centrifugal separation.

The mixture of RNA and DNA oligonucleotides (all T_(m) values below werecalculated for 50 mM NaCl) was made by combining the following: RNA₁ =5′-UAUUGCACUUGUCCCGGCCUG-3′ (21 bases, SEQ ID NO:1) T_(m) 46° C. RNA₂ =5′-GAGACCCAGUAGCCAGAUGUAGCUU-3′ (25 bases, SEQ ID NO:2) T_(m) 61° C.;RNA_(2-COMPL)(“RNA_(2′)”) = 5′-AAGCUACAUCUGGCUACUGGGUCUC-3′ 25 b (SEQ IDNO:3) T_(m) 62° C.; DNA_(A) = 5′-AGCTGTCTAGGTGACACGCTAGAGTACTCGAGCTA-3′(35 bp, SEQ ID NO:4) T_(m) 65° C.; DNA_(A′-COMPL) =5′-TAGCTCGAGTACTCTAGCGTGTCACCTAGACAGCT-3′ (SEQ ID NO:5) T_(m) 65° C.;DNA_(B) = 5′-GTTACACATGCCTACACGCTCCATCATAGG-3′ (30 bases, SEQ ID NO:6)T_(m) 62° C..There was an excess of either one of the complementary sequences (forexample RNA₂ and its complementary sequence, denoted as “RNA_(2-COMPL)”or “RNA₂.”) or the other oligonucleotide, so that one of the singlestranded RNA (25 base), or DNA (35 base) oligonucleotides was present inthe mixture, in addition to the double stranded DNA or double strandedRNA which consisted of the two hybridized complementary sequences.

The initial binding mixture was composed of: 24 ul of theoligonucleotide mix (above), added to 60 ul Lysis Buffer (Promega RNALysis Buffer, including 1% beta mercaptoethanol), which was then addedto 120 ul of Dilution Buffer (Promega RNA Dilution Buffer). All thetubes used in this example were autoclaved 1.5 ml polypropylenemicro-centrifuge tubes. 17 ul aliquots of the above mixture weredispensed into tubes, in triplicate, for each temperature tested.Duplicate samples of “plus zeolite at 21° C.” and duplicate samples of“plus zeolite at 38° C.” were run. Single control samples were runwithout added zeolite for both 21° C. and 38° C. (control solutions wereincubated and centrifuged through nylon membranes under identicalconditions with the “plus zeolite” samples). For the “plus zeolite”tubes, 2.5 ul of zeolite solution (47% wt/vol of 13× zeolite in 2M NaCl,0.1 mM EDTA) was added to each of the tubes with the oligonucleotidemixture. All samples were mixed and held at 21° C. for 5 minutes, thentwo “plus zeolite” and one “no zeolite added” control tube wereincubated at 38° C. (note that this is well below the T_(m) for theabove mixture components) for 15 minutes. The remaining three sampleswere incubated at 21° C. for 15 minutes. Then the samples weretransferred to Corning nylon 0.22μ columns, and centrifuged at 11,000×gfor 30 seconds. Columns were transferred to fresh tubes. The zeoliteparticles were then washed in 19 ul of a Lysis/Dilution mixture[composed of 100 ul Lysis Buffer, combined with 200 ul Dilution Buffer]as a method of washing the zeolite particles and nylon membrane. Theseparation process was repeated and the columns placed in fresh tubes.The zeolite particles were then eluted in 19 ul of nuclease free waterfor 5 minutes at 21° C. The separation process was repeated and thecolumns placed in fresh tubes. The elution process was repeated a secondtime. The zeolite particles were then resuspended in 39 ul of nucleasefree water for 5 minutes at 21° C. The resulting solutions (includingzeolite particles in elution 3) were removed from the column, and placedin fresh tubes. The samples were run on 15% acrylamide gels, and thesamples stored at −20° C.

For each of the above tubes, 5 ul was removed and loaded per lane onto a15% acrylamide formamide gel, and separated by electrophoresis, exceptthat 10 ul was used in the final elution of the zeolite particles (as 39ul elutions were performed in the third elution, compared to 19 ul forelutions 1 and 2). The elution samples required about 2 hours at 80volts in TBE buffer and the flowthrough samples containing Lysis Bufferand Dilution Buffer required about 7 hours at 40 volts in TBE buffer(due to the significant salt effects on the separation).

The resulting gel scans shown in FIGS. 20A and 20C demonstrated thatboth double stranded DNA (top band, 35 base pairs) and single strandedDNA (third band down, 35 bases) were bound to zeolite 13X at both 21° C.and 38° C. In contrast, FIGS. 20A and 20C also showed that both doublestranded RNA (second band down, 25 base pairs) and single stranded RNA(25 bases and 21 bases) have much lower binding to zeolite at both 21°C. and 38° C. compared to the binding of DNA. The bound double strandedDNA and single stranded DNA was shown to elute in FIGS. 20B and 20C.While some double stranded RNA was eluted from the zeolite, this wasmuch less than the amount of double stranded RNA that did not bind tothe zeolite in the flowthrough.

It was also noted that the “no zeolite” samples showed some binding ofRNA, which may have been due to the nylon membrane that was used in theseparation process. Similarly the first and second elutions of the “nozeolite” samples showed some RNA being eluted, which may have been dueto the nylon membrane used in the column separation. This suggests thatthe double stranded RNA seen in the “+zeolite” samples may largely bedue to binding to the nylon membrane, rather than to binding to thezeolite. Moreover, the “+zeolite” samples shown in elution 3 of FIGS.20B and 20D did not show visible eluted RNA, which suggests that the RNAseen to elute in the first and second elutions was likely due to itsbinding to the nylon membrane. Once the zeolite was separated from thenylon membrane in the third elution, no RNA was shown to elute from thezeolite. In contrast, both double stranded DNA and single stranded DNAwere eluted in the third elution from the zeolite when it was separatedfrom the nylon membrane (FIGS. 20B and 20D).

Comparing both the binding (FIGS. 20A and 20C) and elutions (FIGS. 20Band 20D), it was apparent that the heating of samples from 21° C. to 38°C. increased the binding of double stranded DNA to the zeolite.Comparing the flowthrough bands of 21° C. (FIG. 20A) to those of 38° C.(FIG. 20C), double stranded DNA was more completely bound to the zeoliteat 38° C. The amount of double stranded RNA remaining unbound wasrelatively similar between 21° C. and 38° C., which is consistent withthe idea that the observed background binding of RNA to the nylonmembrane was not affected much by the increased temperature.

Example 24 Zeolite Binding of Genomic DNA and Oligonucleotides, andElution Therefrom

This example describes the binding of zeolite to a mixture of: genomicDNA (Promega catalog # G304A, 90% of which is 50,000 base pairs (bp) orlarger), double stranded 35 bp DNA, single stranded 35 base DNA, singlestranded 30 base DNA, 25 bp double stranded RNA, 25 base single strandedRNA and 20 base single stranded RNA. The nucleic acid bound to thezeolite particles was separated from the remaining aqueous phase byeither (A) forming a zeolite pellet by centrifugation, or (B) placingthe zeolite particles in a Corning 0.22μ nylon membrane Spin-X column(Corning catalog # 8169, Corning, N.Y.) followed by centrifugalseparation, or (C) magnetic separation of zeolite coated Fe₂O₃particles.

The mixture of RNA and DNA oligonucleotides was made by combining thefollowing: RNA₁ = 5′-AGACCCAGUAGCCAGAUGUA-3′ (20 bases, SEQ ID NO:7)T_(m) 56° C. RNA₂ = 5′-GAGACCCAGUAGCCAGAUGUAGCUU-3′ (25 bases, SEQ IDNO:2) T_(m) 61° C.; RNA_(2-COMPL) = 5′-AAGCUACAUCUGGCUACUGGGUCUC-3′ (SEQID NO:3) T_(m) 62° C.; DNA_(A) =5′-AGCTGTCTAGGTGACACGCTAGAGTACTCGAGCTA-3′ (35 bp, SEQ ID NO:4)T_(m) 65° C.; DNA_(A-COMPL) = 5′-TAGCTCGAGTACTCTAGCGTGTCACCTAGACAGCT-3′(SEQ ID NO:5) T_(m) 65° C.; DNA_(B) =5′-GTTACACATGCCTACACGCTCCATCATAGG-3′ (30 bases, SEQ ID NO:6)T_(m) 62° C..

There was an excess of either one of the complementary sequences(−_(COMPL)) or the other, so that one of the single stranded RNA (25base), or DNA (35 base) oligonucleotides was present in the mixture inaddition to the double stranded molecules. Hybridization between the R₁20 base sequence and the complementary R₂ 25 base sequence generatedmolecules that were 20 base pairs with a 5 base single stranded overhang(third band from the top in the oligo ladder). Because it is partlydouble stranded and partly single stranded, it is listed as “20 bp/5b”(20 bases double stranded and 5 bases single stranded) on the gel scanimages although it is referred to here as double stranded for purposesof brevity.

The initial binding mixture was composed of: 24 ul of theoligonucleotide mixture (above) added to 60 ul RNA Lysis Buffer (Promegacat# Z3051, including 1% beta mercaptoethanol), which was then added to120 ul of Dilution Buffer. All the tubes used in this example wereautoclaved 1.5 ml polypropylene micro-centrifuge tubes. 17 ul aliquotsof the above mixture were dispensed into tubes, in triplicate, for eachzeolite condition tested (“pellet”, “spin column”, and “magneticparticle”). For all but the “initial mix” tubes, 2.5 ul of zeolitesolution (47% wt/vol of 13× zeolite in 2M NaCl, 0.1 mM EDTA) for“pellet” and “spin column” and 2.5 ul of 0.1% (wt/vol) Fe₂O₃-zeolite 3Amade as described in Example 13 was added to each of the tubes with theinitial mixture of human genomic DNA plus oligonucleotides. All sampleswere mixed and held at 21° C. for 5 minutes, then 70° C. for 5 minutes.The “spin column” samples were transferred to Corning nylon 0.22μcolumns (above), and centrifuged at 11,000×g for 1 minute, along withthe “pellet” sample tubes. The “magnetic zeolite” samples were placed ona magnetic rack and the supernatants removed to clean tubes and storedat −20° C. The pellet supernatants and column flowthroughs weretransferred to fresh tubes and stored at −20° C. The zeolite particleswere then resuspended in 17 ul of ⅓ Lysis Buffer (above) and ⅔ DilutionBuffer (above). The separation process was repeated and the resultingsolutions stored in fresh tubes at −20° C.

The zeolite particles were then resuspended in 17 ul of nuclease freewater for 5 minutes at 21° C. The separation process was repeated andthe resulting solutions stored in fresh tubes at −20° C. The elutionprocess was repeated a second time. The zeolite particles were thenresuspended in 30 ul of nuclease free water for 5 minutes at 21° C. Theresulting solutions (including zeolite particles) were stored at −20° C.

For each of the above tubes, 5 ul was removed and loaded per lane onto a14% acrylamide formamide gel, and separated by electrophoresis, exceptthat 30 ul was used in the final elution of the zeolite particles in thenylon 0.22μ column to facilitate resuspension of the zeolite particles,and 10 ul of this elution was loaded per well. The elutions requiredabout 90 minutes at 100 volts in TBE buffer and the samples containingLysis Buffer and Dilution Buffer (as above) required about 6 hours at 40volts (due to the significant salt effects on the separation).

The resulting gel scans shown in FIGS. 21A and 21C have shown that bothdouble stranded (top band) and single stranded DNA (fourth and fifthbands down) bind to zeolite 13×, but the two double stranded RNA bands(second and third from the top) substantially remained unbound. As notedin Example 23, the column based separations (seen in FIG. 21A) haveintroduced the binding of RNA to the nylon membrane in the column, butthis variable is not present in the “pelleted” samples, as shown in FIG.21C. When looking at the third elutions shown in FIGS. 21B and 21D(lanes 7, 8, and 9 in both gels) the DNA bands (both double stranded andthe two single stranded bands) were clearly visible in all 6 samples,but none of the 6 samples showed visible RNA bands (second and thirdfrom top). This data has been consistent with the data of Example 23,showing that (a) both double stranded DNA and single stranded DNA arebound to, and eluted from, zeolite 13X, and (b) RNA is not substantiallybound to zeolite 13×.

FIGS. 21A through 21D have shown that genomic DNA was bound to zeolite13X, but has not shown any visible bands in any of the elutions. Theelutions showed no visible genomic DNA, even in the third set ofelutions where the particles were also included in the sample loadedonto the gel. While not necessary to understand or practice the presentinvention, one explanation for this may be that the large size of thegenomic DNA (90% is 50 kb or larger) would have bound in multiple sitesto the zeolite, and thus would have difficulty eluting from the zeolitesurface. This property would further enable the separation of RNA fromthe higher molecular weight genomic DNA found in homogenized tissuelysates.

FIGS. 21E and 21F have shown that paramagnetic zeolite 3A particles bindboth DNA and RNA, in contrast to the properties of zeolite 13X, notedabove.

Example 25 Determination of Genomic DNA and Specific mRNA Content ofTotal RNA Samples

This example describes the quantitation of genomic DNA contamination intotal RNA samples by quantitative PCR (qPCR) and the quantitation ofspecific mRNAs by quantitative RT-PCR (qRT-PCR). Promega's Plexor™ qPCRSystem (Cat.# A4011) and Plexor™ One-Step qRT-PCR System (Cat.# A4021)were used. The sensitivity of the Plexor™ technology allowed accuratequantitation of as few as ten copies of a single haploid gene by qPCR.The Plexor™ qPCR reactions were set up on ice as described in thePlexor™ qPCR System Technical Manual (Part# TM262, Promega Corporation,Madison, Wis.). A reaction mix was prepared, composed of 12.5 μl of 2×Plexor™ Master Mix, 1.0 μl of a 25× Plexor™ primer pair mix (5 μM ofeach primer) and Nuclease-Free Water to a final volume of 20 μl perreaction. The final concentration of each primer in the assay was 200nM. Twenty μl of reaction mix was distributed to individual wells of a96-well Optical Reaction Plate (Applied Biosystems, Foster City,Calif.). Five μl of each prepared sample or standard was added to thewells containing reaction mix for a total volume of 25 μl. Each sampleand standard was analyzed in triplicate. The optical plate was sealedwith an Optical Adhesive Cover. The sealed plate was briefly centrifugedto collect the reactions to the bottom of the wells. Primers thatamplified the human thyroid peroxidase (TPOX) short tandem repeat (STR)locus were used for specific quantitation of genomic DNA in the presenceof total RNA. The forward primer (5′FAM iso-dC-GTCCTTGTCAGCGTTTATTT; SEQID NO:8) was labeled with fluorescein (FAM) and contained a nucleotideanalog, methylisocytosine (iso-dC). The reverse primer (5′HO-CCCAGAACCGTCGACTG; SEQ ID NO:9) was unlabelled. Primers weresynthesized by EraGen Biosciences (Madison, Wis.) and were diluted inPromega's MOPS/EDTA Buffer (Cat.# Y5101), protected from light.Promega's Human Genomic DNA (Cat.# G3041) standard curve was used forquantitation. Human Genomic DNA was diluted in MOPS/EDTA Buffer to theequivalent of 10,000, 1,000, 100 and 10 haploid genome copies perreaction. The standard curve was based on the assumption that 33 ng ofhuman genomic DNA was equivalent to 10,000 haploid genome copies. Thatassumption was based on estimates of 6.6 pg of genomic DNA per humandiploid cell and a human genome size of 2.9 Gb. The total RNA sampleswere diluted to 400 ng/μl in Promega's Nuclease-Free Water, based onabsorbance at 260 nm. The samples were then diluted to 20 ng/μl inMOPS/EDTA Buffer. One hundred ng of each total RNA sample was analyzedper reaction. The Plexor™ qPCR amplifications were run on an AppliedBiosystems 7500 Real-Time PCR System instrument (Applied Biosystems,Foster City, Calif.) as described in the Plexor™ Systems InstrumentSetup and Data Analysis for the Applied Biosystems 7300 and 7500Real-Time PCR Systems Technical Manual (Part #TM265, PromegaCorporation, Madison, Wis.). Data analysis was performed using Plexor™Analysis Software (v.1.1.4, Java VM Version 1.4.2_(—)04-b05, PromegaCorporation, Madison, Wis., © EraGen Biosciences, Madison, Wis.).

FIG. 22A shows the Plexor™ TPOX qPCR standard curve, generated from adilution series of Human Genomic DNA. FIG. 22B shows the results ofPlexor™ qPCR analysis of the HEK293T 1E8 1MIX and 2MIX total RNA samplesthat were generated by Example 1. In that example, total RNA wasisolated from 1×10⁸ (1E8) human HEK293T cells per isolation. The 1E81MIX 1, 1MIX 2, 2MIX 1 and 2MIX 2 samples (designated “1 MIX” and “2MIX”for sample groups) were shown in black and the DNA standards were shownin grey. The genomic DNA amounts were 10,000, 1,000, 100 and 10 haploidgenome copies, from left to right across the graph indicated by arrows.The Plexor™ technology was based on quenching of a fluorescent primerduring PCR or RT-PCR amplification (e.g., Frackman, S., et al. (2006)Promega Notes 92, 10-13). The initial reactions showed high levels offluorescence from the fluorescently labeled primer. For the DNAstandards, the fluorescent signal decreased proportionally to the amountof template in the reaction. This was due to the specific incorporationof a quencher from the Plexor™ reaction mix, dabcyl-isoguanine(dabcyl-iso-dGTP). The dabcyl quencher was incorporated opposite theiso-dC residue of the labeled primer during amplification, resulting inquenching of the fluorescent signal. As the PCR product accumulatedexponentially, the signal decreased proportionally. The earlyexponential phase was analyzed to provide the most accuratequantitation. The fluorescence of each sample (relative fluorescenceunits (RFU)) was measured at each PCR cycle by the Real-Time PCR Systeminstrument. A plot of the fluorescence vs. cycle number was generatedfor each sample. The threshold was calculated, based on the initialbackground fluorescence. The calculated base line was plotted as ahorizontal line, indicated by an arrow in FIG. 22B. When eachfluorescent signal decreased below the threshold, the sample wasassigned the corresponding cycle threshold (C_(t)) value. A plot of theC_(t) value vs. the initial template concentration of the Human GenomicDNA standards generated a standard curve that was used to quantitate theunknowns. Higher template amounts produced lower C_(t) values, due toearly quenching. Lower template amounts produced progressively higherC_(t) values. The absence of a detectable amount of template resulted ina curve with high fluorescence that usually did not cross the threshold,unless non-specific amplification products were generated. “No TemplateControl” reactions (NTC) were included as a negative control to detectbackground DNA contamination in the reaction mix. The NTC reactionsconsisted of 5 μl of MOPS/EDTA Buffer added to 20 μl of reaction mix, inplace of template. The NTC reactions displayed high fluorescence with nospecific amplification products or C_(t) values generated (not shown).This indicated that no background DNA was detected. The 1 MIX 1, 1 MIX2, 2MIX 1 and 2MIX 2 amplification curves were similar to the NTCamplification curves. They displayed high fluorescence, with no specificamplification products or C_(t) values. No genomic DNA was detected inthe 1 MIX or 2MIX samples when 100 ng of total RNA was amplified perreaction. Of note, an outlier occurred in one triplicate reaction andwas labeled “NS” for non-specific amplification. This was based on thelower T_(m), as described below.

Quenching of the fluorescently labeled PCR products was reversible byheat denaturation. The Plexor™ technology included an optional thermalmelt curve to determine the melting temperature of the PCR products. Asthe completed reactions were heated and then cooled, followingamplification, the fluorescent signals peaked at the melting temperature(T_(m)). This was due to denaturation, followed by reannealing, of thetwo DNA strands. As the two strands separated, the fluorescenceincreased, due to the quencher no longer being adjacent to thefluorescent label. This property reversed when the strands cooled andreannealed. Determining the T_(m) ensured that changes in fluorescencewere due to specific amplification, rather than the production of primerdimers or non-specific amplification products. Specific amplificationwas indicated by a peak of fluorescence, with a clearly defined T_(m).Melting curves that showed broadening, multiple peaks or peaks with analtered T_(m) indicated non-specific amplification products were present(Frackman, S., et al. (2006) Promega Notes 92, 10-13). This resultedfrom differences in the length and GC content of the non-specificproducts. The melting curves for the Plexor™ qPCR reactions were shownas an inset to FIG. 22B.

FIG. 22C shows a comparative analysis of total RNA samples isolatedusing Promega's SV Total RNA Isolation System (Cat.# Z3100). Thesesamples were analyzed in the same experiment as the samples in FIG. 22B.They were shown as a separate figure, in order to distinguish betweenthe samples sets without the use of color. The same standard curve andNTC samples were used for both figures. Total RNA was isolated from5×10⁶ human HEK293T cells per isolation, with or without DNase treatment(n=4 each). The samples were prepared as in FIG. 22B for qPCR. The samestandard curve and NTC samples were used. The SV +/− DNase samples wereshown in black and the DNA standards were shown in grey. The genomic DNAamounts were 10,000, 1,000, 100 and 10 haploid genome copies, from leftto right across the graph as in FIG. 22B. The standard protocol in theSV Total RNA Isolation System Technical Manual (Promega Corporation,Madison, Wis.) included an on-membrane DNase digestion that removedgenomic DNA during the purification process. These samples, designated“SV+DNase,” produced specific amplification products with high C_(t)values, indicating residual genomic DNA contamination. The SV+DNasevalues fell below the 10 copy DNA standards, indicating less than 10copies of genomic DNA contamination per 100 ng of total RNA. Note thatone of the triplicate reactions did not give a C_(t) value and isvisible as a line above the threshold. The “SV-DNase” samples werepurified without DNase treatment. They gave significantly lower C_(t)values, indicating higher levels of genomic DNA contamination. Themelting curves were shown as an inset. The results showed a clear effectof DNase treatment on the amount of genomic DNA contamination. Theintegrated DNase treatment dramatically reduced genomic DNAcontamination in the SV+DNase samples, compared to the SV-DNase samples.Without DNase treatment, genomic DNA co-purified with the total RNA.

The results in FIG. 22C demonstrated that DNase treatment was notcompletely effective at removing all of the genomic DNA from thesamples. In contrast, the 1MIX and 2MIX total RNA samples, purifiedusing Molecular Sieves, type 13X (zeolite) and an acidic dilutionbuffer, showed complete removal of genomic DNA in FIG. 22B. The resultswere summarized in Table 12. Average Genomic # HEK293T DNA ContaminationCells per per 100 ng Total RNA Total RNA Standard Sample ID Isolation (#TPOX Copies) Deviation 1E8 1MIX 1 1 × 10⁸ 0 NA 1E8 1MIX 2 1 × 10⁸ 0 NA1E8 2MIX 1 1 × 10⁸ 0 NA 1E8 2MIX 2 1 × 10⁸ 0 NA SV + DNase 1 5 × 10⁶ 2.91.0 SV + DNase 2 5 × 10⁶ 3.4 0.5 SV + DNase 3 5 × 10⁶ 4.5 2.9 SV + DNase4 5 × 10⁶ 7.0 2.3 SV − DNase 1 5 × 10⁶ 2,362.6 96.6 SV − DNase 2 5 × 10⁶2,805.8 359.9 SV − DNase 3 5 × 10⁶ 1,911.9 205.4 SV − DNase 4 5 × 10⁶2,542.6 215.2 1MIX/2MIX Samples 1 × 10⁸ 0 NA SV + DNase Samples 5 × 10⁶4.6 2.4 SV − DNase Samples 5 × 10⁶ 2,405.7 396.4NA = not applicable

FIGS. 22D through 22G show the results of qRT-PCR using the 1E8 1MIX and2MIX samples. The Plexor™ One-Step qRT-PCR System was used to quantitateGAPDH (glyceraldehyde-3-phosphate-dehydrogenase) and EDNRB (endothelinreceptor type B) mRNAs in 100 ng of total RNA. The total RNA sampleswere prepared in FIG. 22B. The Plexor™ qRT-PCR amplifications weremultiplexed and set up on ice as described in the Plexor™ One-StepqRT-PCR System Technical Manual (Promega Corporation, Madison, Wis.).Two different fluorophores were used. The GAPDH reactions were labeledwith JOE and the EDNRB reactions were labeled with FAM. The GAPDH primersequences were: 5′ JOE-iso-dC-AGCCGAGCCACATCG (primer 1, JOE-labeled;SEQ ID NO: 10) and 5′ HO-GACCAGGCGCCCAATAC (primer 2, unlabeled; SEQ IDNO: 11). The EDNRB primer sequences were:5′FAM-iso-dC-TCCCAATATCTTGATCGCCAGCT (primer 1, FAM-labeled; SEQ IDNO:12) and 5′ HO-TCTCAGCTCCAAATGGCCAGT (primer 2, unlabeled SEQ IDNO:13). Primers were synthesized by EraGen Biosciences (Madison, Wis.)and were diluted in MOPS/EDTA Buffer, protected from light. A reactionmix was prepared, composed of 12.5 μl of 2× Plexor™ Master Mix, 0.5 μlof RNasin® Plus RNase Inhibitor, 1.0 μl of a 25× Plexor™ primer pair mix(5 μM of each primer), 0.0625 μl of ImProm-II™ Reverse Transcriptase andNuclease-Free Water to a final volume of 20 μl per reaction. The finalconcentration of each primer in the assay was 100 nM for GAPDH and 200nM for EDNRB. Twenty μl of reaction mix was distributed to individualwells of a 96-well Optical Reaction Plate (Applied Biosystems, FosterCity, Calif.). Five μl of each prepared sample or standard was added tothe wells containing reaction mix for a total volume of 25 μl. Eachsample and standard was analyzed in triplicate. The optical plate wassealed with an Optical Adhesive Cover. The sealed plate was brieflycentrifuged to collect the reactions to the bottom of the wells. Eachsample and standard was analyzed in triplicate. For the standard curves,a Universal Human Reference RNA (Stratagene Corporation, La Jolla,Calif.), composed of total RNA from 10 different cell lines, was used.The standards were diluted in cold MOPS/EDTA Buffer to generate adilution series, containing the equivalent of 500 ng, 50 ng, 5 ng and0.5 ng of total RNA standard. The Plexor™ qRT-PCR amplifications wererun on an Applied Biosystems 7500 Real-Time PCR System instrument asdescribed in the Plexor™ Systems Instrument Setup and Data Analysis forthe Applied Biosystems 7300 and 7500 Real-Time PCR Systems TechnicalManual (Promega Corporation, Madison, Wis.). Reactions were prepared ina 96-well Optical Reaction Plate with Barcode, sealed with an OpticalAdhesive Cover. Data analysis was performed using Plexor™ AnalysisSoftware (v.1.1.4, Java VM Version 1.4.2_(—)04-b05, Promega Corporation,Madison, Wis., © EraGen Biosciences, Madison, Wis.). FIGS. 22D and 22Eshow the GAPDH qRT-PCR standard curve and amplification curves,respectively. FIGS. 22F and 22G show the EDNRB qRT-PCR standard curveand amplification curves, respectively. The 1MIX and 2MIX samples wereshown in black and the total RNA standards were shown in grey. The RNAstandard amounts were 500 ng, 50 ng, 5 ng and 0.5 ng of total RNAstandard, from left to right across the graph. The calculated baselinewas plotted as a horizontal line as above. “No Template Control”reactions (NTC) were also run (not shown). The NTC samples showed slightnon-specific amplification around cycle 39 (JOE-GAPDH) and cycles 37-39(FAM-EDNRB), based on their lower Tm's. The insets in FIGS. 22E and 22Gshowed the melting curves. The results demonstrated that the 1 MIX and2MIX total RNA samples contained specific, quantifiable mRNAs that werereproducible between separately isolated samples.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inchemistry and molecular biology or related fields are intended to bewithin the scope of the following claims.

1. A method of generating a purified RNA sample from an initial samplethat comprises DNA and RNA molecules, said method comprising; a)contacting said initial sample with; i) a dilution buffer with an acidicpH, and ii) a nucleic acid binding matrix that preferentially binds DNAmolecules in the presence of said dilution buffer, wherein saidcontacting generates a DNA-bound binding matrix; and b) separating saidDNA-bound binding matrix from said initial sample thereby generating apurified RNA sample comprising a plurality of RNA molecules.
 2. Themethod of claim 1, further comprising step c) exposing said purified RNAsample to a binding component such that an RNA-bound binding componentis generated which comprises a plurality of bound RNA molecules.
 3. Themethod of claim 2, further comprising step d) eluting at least a portionof said bound RNA molecules from said RNA-bound binding member with anelution solution such that a purified RNA preparation is generated,wherein said purified RNA preparation comprises a plurality of elutedRNA molecules.
 4. The method of claim 1, wherein said binding matrix isconfigured to bind both double stranded and single stranded DNAmolecules.
 5. The method of claim 1, wherein said binding matrix isconfigured to not bind double stranded or single stranded RNA molecules.6. The method of claim 5, wherein said initial sample comprises a celllysate, wherein said cell lysate comprises lysed cells, and wherein saidplurality of eluted RNA molecules are present in said purified RNApreparation at a level of at least 5 μg of RNA per 1 million of saidlysed cells present in said sample.
 7. The method of claim 1, whereinsaid purified RNA sample is substantially DNA-free.
 8. The method ofclaim 5, wherein said purified RNA preparation is substantiallyDNA-free.
 9. The method of claim 5, wherein said purified RNA sample orsaid purified RNA preparation contains less than 10 copies of a singlecopy gene per 100 ng of RNA in said initial sample.
 10. The method ofclaim 1, wherein said separating comprises passing said initial samplethrough a clearing column.
 11. The method of claim 1, wherein saidseparating comprises centrifuging said initial sample such that a pelletforms which contains said DNA-bound binding matrix, and separating saidpellet from the remainder of said initial sample.
 12. The method ofclaim 1, wherein said binding matrix comprises zeolites and Fe₂O₃ andsaid separating comprises magnetic separation of said DNA-bound bindingmatrix from said initial sample.
 13. The method of claim 1, wherein saidsample is incubated at a temperature of between 25 and 80 degreesCelsius prior to step b).
 14. The method of claim 1, wherein said sampleis incubated at a temperature of about 70 degrees Celsius prior to stepb).
 15. The method of claim 1, wherein said dilution buffer comprises acitrate buffer, or both a citrate buffer and a chaotropic agent.
 16. Themethod of claim 1, wherein said sample comprises a cell lysate.
 17. Themethod of claim 16, wherein said cell lysate comprises non-nucleic acidcellular debris, and wherein said separating said DNA-bound bindingmatrix serves to remove a substantial proportion of said non-nucleicacid cellular debris from said initial sample.
 18. The method of claim1, wherein said binding matrix comprises binding particles, and saidbinding particles are in a composition comprising a salt solution. 19.The method of claim 1, wherein said binding matrix comprises a membranecoated with binding particles.
 20. The method of claim 19, wherein saidbinding matrix comprises silicon.
 21. The method of claim 1, whereinsaid binding matrix comprises pores, wherein said pores are about 3 Å to10 Å in size.
 22. The method of claim 1, wherein said binding matrixcomprises zeolite particles.
 23. The method of claim 1, wherein saiddilution buffer has a pH of about 5.3 or less.
 24. The method of claim1, wherein said DNA molecules in said sample comprise genomic DNAmolecules.
 25. The method of claim 1, wherein the purified RNA samplecontains less than 0.05% of the mass of DNA present in said initialsample.
 26. A kit for generating a purified RNA sample comprising; a) adilution buffer with an acidic pH; and b) a binding matrix, wherein saidbinding matrix is configured to preferentially bind DNA molecules in thepresence of said dilution buffer.
 27. The kit of claim 26, wherein saidbinding matrix comprises binding particles.
 28. The kit of claim 26,further comprising: a lysis buffer, wherein said lysis buffer comprisesa chaotropic agent.
 29. The kit of claim 28, wherein said lysis bufferfurther comprises a reducing agent.
 30. The kit of claim 26, furthercomprising: a wash solution.
 31. The kit of claim 26, furthercomprising: a clearing column.
 32. The kit of claim 26, furthercomprising: a binding column.
 33. The kit of claim 26, wherein saiddilution buffer comprises a citrate buffer.
 34. The kit of claim 26,wherein said binding matrix comprise zeolite particles.
 35. Acomposition comprising; a) zeolite particles present in an amount thatis 35-55% by weight of said composition; b) a NaCl solution present at aconcentration of about 1-3M c) ethylene diamine tetra-acetic acid (EDTA)present at a concentration of about 0.05 to 1.5 mM.
 36. The compositionof claim 35, wherein said zeolite particles are present in an amountthat is about 45-50% by weight of said composition.
 37. The compositionof claim 35, wherein said NaCl solution is present at a concentration ofabout 2M.
 38. The composition of claim 35, wherein said EDTA is presentat a concentration of about 0.1 mM.
 39. The composition of claim 35,wherein said zeolite particles are configured to preferentially bind DNAin the presence of a buffer with an acidic pH.
 40. The composition ofclaim 35, wherein said zeolite particles comprise Type 13X molecularsieve zeolites,
 41. The composition of claim 35, wherein said zeoliteparticles comprise Type 3A molecular sieve zeolites.